Masitinib demonstrates anti-proliferative and pro-apoptotic activity in primary and metastatic feline injection-site sarcoma cells

Dysregulation of platelet-derived growth factor receptor (PDGFR) may play a role in feline injection-site sarcoma (ISS) cell growth and viability. Masitinib, a tyrosine kinase inhibitor approved for treatment of canine mast cell tumours, is highly selective for the PDGFR signalling pathway and may offer a new therapeutic approach for this disease. The in vitro effects of masitinib on growth, apoptosis and PDGFR signalling in two novel ISS cell lines were investigated. PDGFR expression was confirmed by Western blot in cell lines derived from a primary ISS tumour (JB) and a corresponding, histologically confirmed ISS lung metastasis (JBLM). Masitinib inhibited cell growth and PDGFR phosphorylation in both cell lines. Higher drug concentrations were required to inhibit growth than to modulate ligand-induced autophosphorylation of PDGFR. These in vitro data suggest that masitinib displays activity against both primary and metastatic ISS cell line and may aid in the clinical management of ISS.     

The tyrosine kinase inhibitor sorafenib decreases cell number and induces apoptosis in a canine osteosarcoma cell line

Canine osteosarcoma, an aggressive cancer with early distant metastasis, shows still despite good chemotherapy protocols poor long term survival. The aim of our study was to determine whether sorafenib, a novel multikinase inhibitor, has any effect on D-17 canine osteosarcoma cells.A cell proliferation kit was used for detecting surviving cells after treatment for 72 h with sorafenib or carboplatin or their combination. A significant decrease of neoplastic cells was observed after incubation with 0.5-16 μM sorafenib or with 80-640 μM carboplatin. Using immunocytochemistry for activated caspase 3 to evaluate apoptosis, we found significantly more positive cells in the sorafenib treated groups. Paradoxically, expression of the nuclear proliferation marker Ki-67 was also significantly higher in sorafenib treated cells. The drug sorafenib showed potent antitumour activity against D-17 canine osteosarcoma cells in vitro, suggesting a potential as a therapeutic tool in the treatment of bone cancer in dogs.     

Pharmacokinetic and pharmacodynamic interactions of tolfenamic acid and marbofloxacin in goats

Pharmacokinetic and pharmacodynamic properties in goats of the non-steroidal anti-inflammatory drug tolfenamic acid (TA), administered both alone and in combination with the fluoroquinolone marbofloxacin (MB), were established in a tissue cage model of acute inflammation. Both drugs were injected intramuscularly at a dose rate of 2 mg kg⁻¹. After administration of TA alone and TA + MB pharmacokinetic parameters of TA (mean values) were C_max = 1.635 and 1.125 μg ml⁻¹, AUC = 6.451 and 3.967 μg h ml⁻¹, t_1/2K₁₀ = 2.618 and 2.291 h, Vdarea/F = 1.390 and 1.725 L kg⁻¹, and ClB/F = 0.386 and 0.552 L kg⁻¹ h⁻¹, respectively. These differences were not statistically significant. Tolfenamic acid inhibited prostaglandin (PG)E₂ synthesis in vivo in inflammatory exudate by 53-86% for up to 48 h after both TA treatments. Inhibition of synthesis of serum thromboxane (Tx)B₂ ex vivo ranged from 16% to 66% up to 12 h after both TA and TA + MB, with no significant differences between the two treatments.From the pharmacokinetic and eicosanoid inhibition data for TA, pharmacodynamic parameters after dosing with TA alone for serum TxB₂ and exudate PGE₂ expressing efficacy (E_max = 69.4 and 89.7%), potency (IC₅₀ = 0.717 and 0.073 μg ml⁻¹), sensitivity (N = 3.413 and 1.180) and equilibration time (t_1/2K_e0 = 0.702 and 16.52 h), respectively, were determined by PK-PD modeling using an effect compartment model. In this model TA was a preferential inhibitor of COX-2 (COX-1:COX-2 IC₅₀ ratio = 12:1). Tolfenamic acid, both alone and co-administered with MB, did not affect leucocyte numbers in exudate, transudate or blood. Compared to placebo significant attenuation of skin temperature rise over inflamed tissue cages was obtained after administration of TA and TA + MB with no significant differences between the two treatments. Marbofloxacin alone did not significantly affect serum TxB₂ and exudate PGE₂ concentrations or rise in skin temperature over exudate tissue cages. These data provide a basis for the rational use of TA in combination with MB in goat medicine.     

Effect of gentle stroking and vocalization on behaviour,mucosal immunity and upper respiratory disease in anxious shelter cats

Emotional, behavioural, and health benefits of gentle stroking and vocalizations, otherwise known as gentling, have been documented for several species, but little is known about the effect of gentling on cats in stressful situations. In this study, 139 cats rated as anxious upon admission to an animal shelter were allocated to either a Gentled or Control group. Cats were gentled four times daily for 10 min over a period of 10 days, with the aid of a tool for cats that were too aggressive to handle. The cats’ mood, or persistent emotional state, was rated daily for 10 d as Anxious, Frustrated or Content. Gentled cats were less likely to have negatively valenced moods (Anxious or Frustrated) than Control cats (Incidence Rate Ratio [IRR] = 0.61 CI 0.42–0.88, P = 0.007). Total secretory immunoglobulin A (S-IgA) was quantified from faeces by enzyme-linked immunosorbent assay. Gentled cats had increased S-IgA (6.9 ± 0.7 log_e μg/g) compared to Control cats (5.9 ± 0.5 log_e μg/g) (P < 0.0001). Within the Gentled group of cats, S-IgA values were higher for cats that responded positively to gentling (7.03 ± 0.6, log_e μg/g), compared with those that responded negatively (6.14 ± 0.8, log_e μg/g). Combined conjunctival and oropharyngeal swab specimens were tested by quantitative real-time polymerase chain reaction (rPCR) for feline herpesvirus type 1 (FHV-1), feline calicivirus (FCV), Mycoplasma felis, Chlamydophila felis, and Bordetella bronchiseptica. There was a significant increase in shedding over time in Control cats (23%, 35%, 52% on days 1, 4 and 10, respectively), but not in gentled cats (32%, 26%, 30% on days 1, 4 and 10, respectively) (P = 0.001). Onset of upper respiratory disease was determined by veterinary staff based on clinical signs, in particular ocular and/or nasal discharge. Control cats were 2.4 (CI: 1.35–4.15) times more likely to develop upper respiratory disease over time than gentled cats (P < 0.0001). It is concluded that gentling anxious cats in animal shelters can induce positive affect (contentment), increase production of S-IgA, and reduce the incidence of upper respiratory disease.     

Henneguya corruscans n. sp. (Myxozoa,Myxosporea, Myxobolidae), a parasite of Pseudoplatystoma corruscans (Osteichthyes,Pimelodidae) from the Paraná River,Brazil: A morphological and morphometric study

This paper describes the parasite Henneguya corruscans n. sp. which infects the gills of Pseudoplatystoma corruscans Spix and Agassiz, 1829 found in the Paraná River, Brazil. The parasites belong to the interlamellar-epithelial type as defined by Molnár (2002) [Molnár, K., 2002. Site preference of fish myxosporeans in the gills. Dis. Aquat. Org. 48, 197–207]. The spores examined had thin, smooth walls with symmetric valves; the total length of the spores was 27.6 (25–29) μm. The spore body was ellipsoidal in frontal view and biconvex in lateral view and they measured 14.3 (13–15) μm long by 5 μm wide and 4 μm in thickness. The polar capsules were small and elongated, equally sized, with a rounded posterior extremity and tapering anteriorly, and they corresponded more or less the half the length of the spore body; they were 6.8 (6–7) μm long by 2 μm wide, and the polar filament formed 5–6 coils obliquely to the axis of the polar capsule. The tail was 13.7 (12–15) μm long and bifurcated shortly after the end of the spore body. The importance of the infection for the farming of P. corruscans is discussed.     

Antimicrobial susceptibility testing of Spanish field isolates of Brachyspira hyodysenteriae

This study is the first conducted in Spain to evaluate antimicrobial susceptibility of field isolates of Brachyspira hyodysenteriae. One hundred and eight isolates of the bacterium, recovered from different Spanish swine farms between 2000 and 2007, were investigated. The minimum inhibitory concentrations (MIC) of erythromycin, tylosin, tiamulin, valnemulin, clindamycin and lincomycin were determined using a broth microdilution technique. Most of the isolates showed poor susceptibility to erythromycin (MIC₉₀ > 256 μg/ml), tylosin (MIC₉₀ > 256 μg/ml), clindamycin (MIC₉₀ > 4 μg/ml) and lincomycin (MIC₉₀ = 128 μg/ml). Reduced susceptibility to tiamulin and valnemulin was observed with a MIC > 2 μg/ml in 17.6% and 7.41% of the B. hyodysenteriae isolates, respectively. Moreover, a survival analysis permitted the detection of an increasing trend in the MIC values for almost all the antimicrobials used in the treatment of swine dysentery when comparing recent isolates (from 2006 to 2007) with those recovered in earlier years (between 2000 and 2004).     

Characteristics of prolactin-releasing response to salsolinol (SAL) and thyrotropin-releasing hormone (TRH) in ruminants

The secretion of prolactin (PRL) is stimulated by thyrotropin-releasing hormone (TRH), and inhibited by dopamine (DA). However, we have recently demonstrated that salsolinol (SAL), a DA-derived endogenous compound, is able to stimulate the release of PRL in ruminants. The aims of the present study were to compare the characteristics of the PRL-releasing response to SAL and TRH, and examine the relation between the effects that SAL and DA exert on the secretion of PRL in ruminants in vivo and in vitro. Three consecutive intravenous (i.v.) injections of SAL (5 mg/kg body weight (b.w.): 19.2 μmol/kg b.w.) or TRH (1 μg/kg b.w.: 2.8 nmol/kg b.w.) at 2-h intervals increased plasma PRL levels after each injection in goats (P < 0.05); however, the responses to SAL were different from those to TRH. There were no significant differences in each peak value between the groups. The rate of decrease in PRL levels following the peak was attenuated in SAL-treated compare to TRH-treated animals (P < 0.05). PRL-releasing responses to SAL were similar to those to sulpiride (a DA receptor antagonist, 0.1 mg/kg b.w.: 293.3 nmol/kg b.w.). In cultured bovine anterior pituitary (AP) cells, TRH (10⁻⁸ M) significantly increased the release of PRL following both 15- and 30-min incubation periods (P < 0.05), but SAL (10⁻⁶ M) did not increase the release during the same periods. DA (10⁻⁶ M) completely blocked the TRH-induced release of PRL for a 2-h incubation period in the AP cells (P < 0.05). Sulpiride (10⁻⁶ M) reversed this inhibitory effect but SAL (10⁻⁶ M) did not have any influence on the action of DA. These results show that the mechanism(s) by which SAL releases PRL is different from the mechanism of action of TRH. Furthermore, they also show that the secretion of PRL is under the inhibitory control of DA, and SAL does not antagonize the DA receptor's action.     

Purification of cattle oviduct specific proteins and their effect on in vitro embryo development

The purpose of this study was to determine the effect of oviduct specific proteins as a media supplement for in vitro embryo development in cattle. The proteins were extracted from oviducts of cows and precipitated by ammonium sulfate (30%, 40%, 50% and 60%) followed by dialysis in 50 mM Tris–HCl (pH 7.0) buffer. The dialyzed proteins were fractionated into acidic, basic and neutral fractions using SP sephadex cation exchange and DEAE sephadex anion exchange column chromatography respectively. Cow oviduct specific proteins (cOSPs) constituting all the extracted proteins were used as media supplement in three different concentrations (10, 50 and 100 μg/ml) for in vitro maturation, fertilization and culture (IVMFC) of cow oocytes. Acidic, basic and neutral (unbound) fractions were also used as media supplement in three different concentrations (10, 30 and 50 μg/ml) for IVMFC. Cumulus oocytes complexes were collected from slaughterhouse ovaries, washed thoroughly and cultured in maturation media for 24 h in 5% CO₂ at 38.5 °C with maximum humidity. In vitro matured oocytes were co-incubated with in vitro capacitated sperm in Fert-BO media at 38.5 °C for 18 h in 5% CO₂. The fertilized oocytes were washed and cultured in embryo development media for cleavage. After 40–42 h cleavage was observed and embryos were put in the replacement media for further development. The cleavage rates (%) for cOSPs were observed as 68.24±2.46, 69.28±2.05, 61.77±0.93 and 42.62±1.31 at concentrations of 0, 10, 50 and 100 μg/ml respectively. Rates of blastocyst stage development were 14.49±3.61, 21.17±2.77, 14.66±1.06 and 11.98±1.84. These results indicate that addition of cOSP at10 μg/ml increased blastocyst formation as compared to other concentrations (0, 50 and 100 μg/ml). Although acidic, basic and neutral fractions seemed to have no major effect on cleavage rate, but both acidic and neutral fraction of oviduct specific proteins improved the cleavage rate at 30 μg/ml concentration and basic fraction improved the blastocyst formation at 10 μg/ml concentration.     

Cytotoxicity of Senecio in macrophages is mediated via its induction of oxidative stress

In Arunachal Pradesh and other sub-Himalayan areas of India, accidental consumption of Senecio plants by yaks is often fatal as the plant contains toxic alkaloids like Seneciophylline. The present investigation was undertaken to demonstrate the pro-oxidant effects of an ethanolic extract of Seneciochrysanthemoides (S-EtOH). S-EtOH impaired viability in macrophages, the IC₅₀ being 13.8 ± 1.11 μg/mL. The effect of S-EtOH (1 μg/mL) on generation of reactive oxygen species (ROS) in macrophages was measured by flow cytometry using 2′,7′-dichlorofluorescein diacetate (H₂DCFDA) where it caused a significant increase in the mean fluorescence channel (MFC) from 8.55 ± 0.03 to 47.32 ± 2.25 (p < 0.001). S-EtOH also effected a 3.8-fold increase in extracellular nitric oxide (NO) generation from 4.90 ± 0.72 μM to 18.79 ± 0.32 μM (p < 0.001), a 2.2-fold increase in intracellular NO production, the MFC increasing from 14.95 ± 0.48 to 33.34 ± 1.66 (p < 0.001), and concomitantly depleted non protein thiols as analyzed by flow cytometry using mercury orange, with a reduction in MFC from 632.5 ± 49.44 to 407.4 ± 12.61 (p < 0.01). Additionally, S-EtOH (14 μg/mL, 24 h) caused apoptosis as evident by increased Annexin V binding and terminal deoxynucleotidyl transferase mediated dUTP DNA nick end labeling. Taken together, the cytotoxicity of S-EtOH can be partly attributed to its capacity to inflict oxidative damage via generation of both reactive oxygen and nitrogen species culminating in apoptosis.     

Risk factors associated with increased mortality of farmed Pacific oysters in Ireland during 2011

The Pacific oyster, Crassostrea gigas, plays a significant role in the aquaculture industry in Ireland. Episodes of increased mortality in C. gigas have been described in many countries, and in Ireland since 2008. The cause of mortality events in C. gigas spat and larvae is suspected to be multifactorial, with ostreid herpesvirus 1 (OsHV-1, in particular OsHV-1 μvar) considered a necessary, but not sufficient, cause. The objectives of the current study were to describe mortality events that occurred in C. gigas in Ireland during the summer of 2011 and to identify any associated environmental, husbandry and oyster endogenous factors. A prospective cohort study was conducted during 2010–2012, involving 80 study batches, located at 24 sites within 17 bays. All 17 bays had previously tested positive for OsHV-1 μvar. All study farmers were initially surveyed to gather relevant data on each study batch, which was then tracked from placement in the bay to first grading. The outcome of interest was cumulative batch-level mortality (%). Environmental data at high and low mortality sites were compared, and a risk factor analysis, using a multiple linear regression mixed effects model, was conducted. Cumulative batch mortality ranged from 2% to 100% (median = 16%, interquartile range: 10–34%). The final multivariable risk factor model indicated that batches imported from French hatcheries had significantly lower mortalities than non-French hatcheries; sites which tested negative for OsHV-1 μvar during the study had significantly lower mortalities than sites which tested positive and mortalities increased with temperature until a peak was reached. There were several differences between the seed stocks from French and non-French hatcheries, including prior OsHV-1 μvar exposure and ploidy. A range of risk factors relating to farm management were also considered, but were not found significant. The relative importance of prior OsHV-1 μvar infection and ploidy will become clearer with ongoing selection towards OsHV-1 μvar resistant oysters. Work is currently underway in Ireland to investigate these factors further, by tracking seed from various hatchery sources which were put to sea in 2012 under similar husbandry and environmental conditions.     

Naproxen in the horse: Pharmacokinetics and side effects in the elderly

It is well-known that old animals show physiologic and/or pathologic variation that could modify the pharmacokinetics of drugs and the related pharmacodynamic response. In order to define the most appropriate therapeutic protocol in old horses, pharmacokinetic profile and safety of naproxen were investigated in horses aged over 18 years after oral administration for 5 days at the dose of 10 mg/kg b.w./day. After the first administration, the maximum concentration (C_max 44.21 ± 9.21 μg/mL) was reached at 2.5 ± 0.58 h post-treatment, the harmonic mean terminal half-life was 6.96 ± 1.73 h, AUC_0–24h was 459.71 ± 69.95 h μg/mL, MRT was 7.44 ± 0.74 h and protein binding was 98.47 ± 2.72%. No drug accumulation occurred with repeated administrations. No clinical and laboratory changes were detected after administration of naproxen. Gastric endoscopies performed after the treatment did not show pathological changes of the gastric mucosa.     

Effects of unsaturated fatty acids on progesterone secretion and selected protein kinases in goat granulosa cells

Previous studies in cattle have shown influences of dietary unsaturated fatty acid (UFA) supplementation on ovarian function. However, it is unclear whether these UFA exert direct or indirect effects on ovarian steroid production or their mechanisms of action. We have recently shown that 5′AMP-activated protein kinase (AMPK) regulates progesterone secretion through mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (MAPK ERK1/2) in rodent granulosa cells. Here, we investigated the effects of 3 UFAs, oleic acid (OA), linoleic acid (LA), and α-linolenic acid (ALA) on progesterone secretion in goat granulosa cells. Finally, we examined the effects of UFAs on MAPK ERK1/2 and AMPK phosphorylation in these granulosa cells. Oleic acid and LA (10 μM each), but not ALA (100 μM), increased progesterone secretion (P < 0.05) in the presence or absence of insulin-like growth factor (IGF)-1 (10^-8 M) or FSH (5 × 10⁻⁸ M). The different AMPK subunits, except for γ3, are present in the goat ovary. Treatment with metformin (10 mM), an activator of AMPK, increased AMPK phosphorylation (P < 0.05) and reduced progesterone secretion by 50% (P < 0.05) in the basal state and in response to IGF-1 or FSH in goat granulosa cells. Oleic acid and LA had no effect on AMPK phosphorylation, whereas they rapidly increased MAPK ERK1/2 phosphorylation (P < 0.05). Finally, U0126, a MAPK ERK1/2 inhibitor, decreased OA- and LA-induced progesterone secretion (P < 0.05), suggesting that these UFAs could stimulate progesterone secretion partly through MAPK ERK1/2 in the absence of IGF-1 and FSH in goat granulosa cells. The involvement of AMPK in this process remains to be demonstrated. Taken together, some fatty acids could improve ovarian steroidogenesis through the MAPK ERK1/2 signaling pathway and, consequently, have beneficial effects on goat fertility.     

Reduction of infectious bursal disease virus replication by shRNAs targeting the VP1 and VP2 genes driven by chicken U6 promoter

Infectious bursal disease virus (IBDV) causes a highly contagious and immunosuppressive disease in young chickens and results in considerable economic losses for the poultry industry. To suppress the replication of IBDV, two short hairpin RNAs (shRNAs) were designed for targeting the VP1 and VP2 genes of IBDV. Recombinant plasmids carrying each shRNA or two shRNAs were constructed based on vector pSilencer2.1-U6 in which the human U6 promoter was replaced with chicken U6 promoter. In chicken embryo fibroblasts, transfection with these shRNA plasmids 24 h before infection with IBDV B87 reduced 50% tissue culture infectious doses (TCID₅₀) from 10^8.75 TCID₅₀/0.1 mL to 10^3.75–10^1.0 TCID₅₀/0.1 mL. In 10-day old specific pathogen-free (SPF) chicken embryos, incubation with a mixture of IBDV B87 and a shRNA plasmid via the allantoic cavity resulted in 100% mortality and high IBDV virus titer in the control group but 25–0% mortality and near normal embryo development in the specific shRNA groups; additionally, IBDV VP1 and VP2 mRNA levels were reduced by 72–95% in the shRNA groups as compared with the control groups. When challenged with a virulent strain IBDV GX8/99, 14-day-old chickens pre-treated with the single shRNA plasmids or the dual shRNA plasmid showed approximately 70% or 90% survival at 5 days post-challenge while those pre-treated with control plasmid or saline had less than 5% survival. The current study suggests that two IBDV shRNAs expressed by a plasmid under chicken U6 promoter could effectively and synergistically reduce IBDV replication in vitro and in vivo.     

Pathogenesis of GIII.2 bovine norovirus,CV186-OH/00/US strain in gnotobiotic calves

The pathogenesis of GIII.2 bovine norovirus (BoNoV) is not well understood. Our study demonstrated persisting diarrhea and prolonged fecal shedding, but with a lack of significant intestinal lesions in gnotobiotic (Gn) calves infected with GIII.2 BoNoV, CV186-OH/00/US strain. Nine 4 to 7-day-old Angus/Jersey crossbred Gn calves were orally inoculated with 10.0–11.9 log₁₀ genomic equivalents (GE)/calf of CV186-OH (n = 7) or mock (n = 2). Calves were euthanized at post-inoculation day (PID) 1 (n = 1) when moderate to severe lethargy was observed and at PIDs 2–6 (n = 4) after lethargy had subsided. Two calves were kept longer term (until PID 30) for monitoring fecal shedding patterns by TaqMan real-time RT-PCR (qRT-PCR). Most infected calves exhibited two clinical signs: (i) acute but persisting diarrhea and (ii) acute moderate to severe lethargy. The two infected calves, followed longer-term, had prolonged fecal viral RNA shedding [peak average titer of 11.8 (±0.2) log₁₀ GE/ml] at least until PID 20. By qRT-PCR, 5 infected calves had low viral RNA titers in serum, ranging from 4.0 to 5.8 log₁₀ GE/ml, at PIDs 1–5, but not (<2.7 log₁₀ GE/ml) at PIDs 6–30. The latter observation coincided with the presence of serum IgG antibody to BoNoV at PIDs 8–30. Collectively, the GIII.2 BoNoV strain CV186-OH induced only mild enteropathogenicity, evident by the lack of significant intestinal lesions, but it led to persisting mild diarrhea and prolonged fecal virus shedding in Gn calves. The prolonged fecal shedding of GIII.2 BoNoV might partially explain how this virus is maintained as endemic infections in cattle.     

Interaction between ascorbic acid and follicle-stimulating hormone maintains follicular viability after long-term in vitro culture of caprine preantral follicles

This study evaluates the effects of ascorbic acid and its interaction with follicle-stimulating hormone (FSH) on the morphology, activation, and in vitro growth of caprine preantral follicles. Ovarian fragments were cultured for 1, 7, or 14 d in minimum essential medium (MEM) containing ascorbic acid (50 or 100 μg/mL), FSH (50 ng/mL), or both of these substances. Ovarian tissue that was either fresh (control) or cultured for 1, 7, or 14 d was processed for histological and ultrastructural evaluation. The results showed that after 14 d of culture, medium supplemented with 50 μg/mL of ascorbic acid alone or combined with FSH showed higher rates of follicular survival compared with MEM. After 7 d of culture, FSH, ascorbic acid at 50 μg/mL with or without FSH, and ascorbic acid at 100 μg/mL increased the percentage of follicular activation compared to fresh control. In addition, FSH alone significantly increased the percentage of growing follicles after 14 d. The combination of 50 μg/mL of ascorbic acid and FSH promoted a significant increase in oocyte and follicular diameter after 7 d of culture. Ultrastructural and fluorescent analysis confirmed the integrity of follicles cultured with 50 μg/mL of ascorbic acid and FSH after 14 d. In conclusion, the combination of 50 μg/mL of ascorbic acid and FSH maintained follicular integrity and promoted follicular activation and growth after long-term in vitro culture of caprine preantral follicles.     

Masitinib as a chemosensitizer of canine tumor cell lines: a proof of concept study

Masitinib, a selective tyrosine kinase inhibitor, has previously been shown to enhance the antiproliferative effects of gemcitabine in human pancreatic cancer, demonstrating potential as a chemosensitizer. This exploratory study investigated the ability of masitinib to sensitize various canine cancer cell lines to doxorubicin, vinblastine, and gemcitabine. Masitinib strongly sensitized histiocytic sarcoma cells to vinblastine (>70-fold reduction in IC(50) at 5 μM masitinib), as well as osteosarcoma and mammary carcinoma cells to gemcitabine (>70-fold reduction at 5-10 μM). In addition, several cell lines were sensitized to doxorubicin (2-10-fold reduction at 10 μM). These data establish proof-of-concept that masitinib in combination with chemotherapeutic agents can generate synergistic growth inhibition in various canine cancers, possibly through chemosensitization. The findings justify further investigation into those combinations that may potentially yield therapeutic benefit.     

In vitro efficacy of doxorubicin and etoposide against a feline injection site sarcoma cell line

Feline injection site sarcoma (ISS) is a locally invasive tumor, in which surgical treatment is frequently combined with radiation or chemotherapy to improve tumor control. The focus of this study was to evaluate the cytotoxic effects of doxorubicin or etoposide on a feline injection site sarcoma cell line (JB) and to assess the impact of combining these drugs on cell death and cell cycle. Both single agent and combination drug administration increased cell death and significantly reduced the number of viable cells. Cells in G0/G1 were significantly reduced while the G2/M fraction was significantly increased following treatment. Collectively, combining doxorubicin and etoposide at the lower EC yielded comparable results to the EC50 of either drug alone in degree of cytotoxicity, level of apoptosis, and % of cells in G2/M. The results of this study indicate that doxorubicin and etoposide alone and in combination differentially alter ISS cell viability and cycle.     

The effects of CO2 gas stunning on meat quality of cattle compared with captive bolt stunning

The aim of this study is to assess the effect of CO₂ gas stunning which has not been conducted until now in comparison with captive bolt stunning on the meat quality of cattle. A total of 40 steers were slaughtered following two stunning processes: CO₂ gas stunning (n=20), exposure at 70% CO₂ gas atmosphere for 140 sec; and captive bolt stunning (CBS, n=20). The slaughtered steers were classified into Group A (620–710 kg) and Group B (720–790 kg) by their live weight. CO₂ gas stunning decreases pH value (p<0.05) but increases lightness (p<0.001) and sarcomere length (p<0.001 and p<0.01 for Group A and Group B, respectively) in all live weight groups. Drip loss was increased with CO₂ gas stunning in Group A (620–710 kg of live weight) (p<0.05), whereas WBSF was decreased with CO₂ gas stunning in Group B (720–790 kg of live weight) (p<0.001). Therefore, CO₂ gas stunning negatively affects muscle pH and water-holding capacity in steers but CO₂ gas stunning can improve the tenderness and lightness compared with captive bolt stunning in cattle.     

Role of ghrelin in regulating rabbit ovarian function and the response to LH and IGF-I

The aim of these in vivo and in vitro studies was to examine the role of ghrelin in the control of plasma hormone concentrations, the proliferation, apoptosis and secretory activity of ovarian granulosa cells and the response of these cells to hormonal treatments. Female rabbits were injected with ghrelin (10 μg/animal/day for one week before ovulation induced by 25 IU PMSG and 0.25 IU LHRH). On the day of ovulation, blood samples were collected and analyzed for concentrations of progesterone (P₄), testosterone (T), estradiol (E₂), estrone-sulphate (ES), insulin-like growth factor I (IGF-I) and leptin (L) by RIA. Some control and ghrelin-treated animals were killed in the periovulatory period, their ovaries were weighed and granulosa cells were isolated and cultured for 2 d. Cell proliferation (expression of PCNA) and apoptosis (expression of TdT) were evaluated by immunocytochemistry and TUNEL respectively. Secretion of P₄, T, E₂, IGF-I, and prostaglandin F (PGF) by granulosa cells cultured with and without LH or IGF-I (1, 10 or 100 ng/ml medium) was assessed by RIA. The remaining control and treated animals were kept until parturition, while the number, viability and body weight of pups were recorded.     

DNA Fragmentation as an Early Indicator of Extender Efficacy: A Preliminary Study

Evaluation of new potential semen extenders is a field of economic and scientific importance, but assessing motility alone may not be sufficient. The objectives of this study were to examine the effect of oxidative damage by short-term exposure to H₂O₂ on stallion sperm motility and DNA fragmentation and to correlate motility to the percentage of DNA damage as assessed by both terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and single-cell gel electrophoresis assays. Motility and DNA fragmentation were determined immediately before cooling (0 hour) and at 24 hours postcooling. The addition of H₂O₂ (300 μM) to the extender had no effect on either total or progressive motility (P > .05). DNA fragmentation as determined by both Comet and TUNEL assays did not differ between 0 hour and those cells stored for 24 hours in the absence of H₂O₂ (P > .05). However, the addition of H₂O₂ to the extender plus incubation for 24 hours resulted in greater total Comet length, tail length, and tail moment as well as an increase in percentage of sperm cells with DNA damage detected by TUNEL compared to 0 hour (P < .05). Motility was not correlated with DNA damaged cells detected by TUNEL or Comet assays (P > .05). In conclusion, although both the Comet assay and TUNEL detected significant DNA fragmentation in cells exposed to H₂O_2, there was not a significant or appreciable effect of H₂O₂ on motility. Therefore, motility alone is likely not the best laboratory assay with which to assess cooled extender efficacy.