peer reviewed | Objective—To culture equine myoblasts from muscle microbiopsy specimens, examine myoblast production of reactive oxygen species (ROS) in conditions of anoxia followed by reoxygenation, and assess the effects of horseradish peroxidase (HRP) and myeloperoxidase (MPO) on ROS production. Animals—5 healthy horses (5 to 15 years old). Procedures—Equine skeletal myoblast cultures were derived from 1 or 2 microbiopsy specimens obtained from a triceps brachii muscle of each horse. Cultured myoblasts were exposed to conditions of anoxia followed by reoxygenation or to conditions of normoxia (control cells). Cell production of ROS in the presence or absence of HRP or MPO was assessed by use of a gas chromatography method, after which cells were treated with a 3,3′-diaminobenzidine chromogen solution to detect peroxidase binding. Results—Equine skeletal myoblasts were successfully cultured from microbiopsy specimens. In response to anoxia and reoxygenation, ROS production of myoblasts increased by 71%, compared with that of control cells. When experiments were performed in the presence of HRP or MPO, ROS production in myoblasts exposed to anoxia and reoxygenation was increased by 228% and 183%, respectively, compared with findings for control cells. Chromogen reaction revealed a close adherence of peroxidases to cells, even after several washes. Conclusions and Clinical Relevance—Results indicated that equine skeletal myoblast cultures can be generated from muscle microbiopsy specimens. Anoxia-reoxygenation– treated myoblasts produced ROS, and production was enhanced in the presence of peroxidases. This experimental model could be used to study the damaging effect of exercise on muscles in athletic horses.

To optimize the most efficient method for porcine in vitro maturation (IVM), we compared the effects of supplementing extracellular vesicles (EVs) derived from porcine follicular fluid (pFF). The cumulus oocyte complexes were grouped into 4 groups with different supplementations as following: pFF (G1), pFF-depleted EVs (G2), EVs (G3) and control (G4) groups. After IVM with different supplementations, maturation rates and the developmental competences of porcine oocytes and blastocyst development were investigated. Additionally, glutathione (GSH) and reactive oxygen species (ROS) levels were measured in mature oocytes. The EVs were isolated and characterized with cryo-TEM and nanoparticle tracking analysis. The pFF significantly affected the maturation rate, whereas the presence of EVs did not show notable difference in the maturation rates. Although there were numerical increases in the measured parameters in EV and pFF-depleted EVs groups, no significant differences were observed between them. The EV group showed similar oocyte maturation rate for both positive and negative control groups. The GSH was not different among the groups, but ROS levels were significantly lower in pFF-supplemented group when compared with other groups with the highest level in the control group. G2 group wasn’t significantly different G1 and G3 group. G3 group wasn’t significantly different from G2 and G4 group. This suggests that EVs in IVM medium which probably effected partially to protect against oxidative stress and potentially enhance the quality of oocytes. This study indicates that the EVs in pFF play a significant role in improving the efficiency of oocyte maturation in porcine.

OBJECTIVE To determine the effects of grape seed extract (GSE), lutein, and fish oil containing omega-3 fatty acids on oxidative stress, migration, proliferation, and viability of lens epithelial cells (LECs). SAMPLE Lens capsules or cultured LECs obtained from canine cadavers. PROCEDURES An antioxidant reductive capacity assay was used to determine reducing capability of each substance. The LECs were cultured and incubated with various substances, including N-acetyl cysteine (NAC), when appropriate, and dimethyl sulfoxide (DMSO) as positive and vehicle control substances, respectively. A dichlorofluorescein assay was used to evaluate reactive oxygen species (ROS) production, and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to determine cell viability. Ex vivo posterior capsule opacification (PCO) was used to evaluate LEC migration and proliferation. RESULTS Antioxidant reductive effects of GSE surpassed those of NAC, lutein, and fish oil containing omega-3 fatty acids. The GSE reduced ROS production in LECs, compared with the DMSO vehicle control, whereas lutein was pro-oxidative. All test substances reduced cell viability. Ex vivo PCO was not altered by GSE, was decreased by lutein, and was increased by fish oil containing omega-3 fatty acids, compared with results for the DMSO vehicle control. CONCLUSIONS AND CLINICAL RELEVANCE Only GSE had significant antioxidant capabilities and reduced ROS production; however, no effect on ex vivo PCO was detected. Fish oil containing omega-3 fatty acids increased ex vivo PCO. No conclusions could be made regarding antioxidant effects of these substances on LECs. These findings suggested that the substances will not decrease PCO.

We defined methods for use of luminol-dependent chemiluminescence (LDCL) and superoxide anion (O2-) production as parameters of the oxidative metabolism of neutrophils isolated from 1.5- to 5-week-old neonatal calves. We determined how variations in blood sample handling, agonist preparation, individual variability, and age of calves influenced the LDCL and O2- responses to certain agonists, and defined concentrations of soluble and particulate agonists that maximally stimulated the oxidative metabolism of bovine neutrophils. Oxidative responses, particularly LDCL, were characterized by marked dayto-day variability, differed greatly within and between calves, were partially age-dependent, and were partially dependent on the individual agonist. Superoxide anion production had substantially less variability. We compared the in vitro oxidative (LDCL and O2-) responses of neutrophils isolated from neonatal calves stimulated by defined concentrations of the agonists-latex, phorbol myristate acetate, calcium ionophore, and opsonized zymosan-with responses to formylated oligopeptides and zymosan-activated serum, and to live, dead, live opsonized, and dead opsonized Pasteurella haemolytica organisms. Opsonization of particulates, pathogenic or nonpathogenic, enhanced the LDCL and O2- responses of stimulated neutrophils although P haemolytica was a less potent stimulant of oxidative functions than were nonbiological agonists. We conclude that the generation of reactive oxygen species by bovine neutrophils in response to P haemolytica is highly dependent on the presence of opsonins and is greatly enhanced in live vs killed bacteria. Futhermore, the in vitro generation of reactive oxygen species, including O2- by stimulated neutrophils, may be of biologic importance if similar events occur in vivo, and could have a major role in the pathogenesis of the acute lung injury associated with pneumonic pasteurellosis.

Chronic cholangiohepatitis (CCH) is a common pathological condition in cats with a guarded prognosis and unknown etiology. Recently, in human medicine, there has been increased interest in enhancing liver defense mechanisms as an effective treatment strategy to control liver diseases that have a poor prognosis. Metallothionein (MT) is a ubiquitous protein, which has been widely researched for its role in liver defense through heavy metal detoxification, neutralization of reactive oxygen species, and liver regeneration. In this study, immunohistochemistry was used to evaluate the role of MT in CCH and hepatocellular regeneration in 34 cats histologically diagnosed with this condition by assessing the correlation between hepatocellular MT and Ki-67 (marker for cellular proliferation) expression with histological parameters of CCH, such as inflammation, fibrosis, and bile duct proliferation. Statistical analysis was performed using the Spearman-rank correlation test. A significant positive correlation was observed between inflammation and the number of MT-positive hepatocytes (r = 0.36, P = 0.03) and MT labelling intensity (r = 0.37, P = 0.03). In 16 of 34 cases (47%) MT labelling intensity was noted to be pronounced towards the centrilobular zone and very weak or absent towards the portal zone. The results suggest that MT is induced in the liver during chronic inflammatory conditions, which could be speculated as a host defensive mechanism to protect the liver from inflammation-mediated liver injury. Therapeutic interventions utilizing MT, therefore, may have a positive effect on cats with chronic cholangiohepatitis.

Resveratrol (RSV, 3,5,4′-trihydroxytrans-stilbene) protects sperm from cryo-induced damage in various animal and human species. In this study, we aimed to assess the effect of dog sperm cryoprotective extender containing RSV on the quality of post-thaw dog sperm. Sperm were collected from 4 Beagles and supplemented with different concentrations of RSV (0, 100, 200, and 400 µM). After thawing, apoptosis index, and reactive oxygen species (ROS) levels were assessed to determine post-thaw sperm quality. Dog sperm cryopreserved with 400 µM RSV showed significant improvement in post-thaw sperm quality with lower apoptosis index and ROS levels (p < 0.05). Our results showed that the supplementation of dog sperm cryoprotective extender with RSV at a concentration of 400 µM improved the post-thaw dog sperm quality in the term of sperm ROS production and apoptosis. In addition, we emphasize the necessity of testing the ROS levels and apoptosis index using flow cytometry to determine the quality of post-thaw semen.

This study investigated oxidative stress biomarkers at 3 different oxygen concentrations in dogs under general anesthesia to determine whether high-concentration oxygen increases oxidative stress. Six healthy beagles were randomly assigned to receive 3 anesthesia protocols (inhalation of 40%, 60%, and 100% oxygen) during 3 hours of general anesthesia with sevoflurane, with at least one week in between each protocol. For each experiment, blood samples were collected at 0, 3, 6, and 24 hours after inhalation of oxygen. Derivatives of reactive oxygen metabolites, biochemical antioxidant potential, superoxide dismutase, and 8-hydroxydeoxyguanosine in the blood did not significantly differ among the 3 groups at any time point. This study is the first comparing high concentrations of oxygen with low concentrations of oxygen for anesthesia in dogs. According to our findings, 100% oxygen may not alter the oxidative stress level in dogs during general anesthesia with sevoflurane for 3 hours.

Objective: To compare mitochondrial complex I and complex IV activity in myocardial mitochondria of clinically normal dogs, clinically normal dogs exposed to inhalation anesthesia, and dogs affected with dilated cardiomyopathy. Sample: Myocardial samples obtained from 21 euthanized dogs (6 clinically normal [control] dogs, 5 clinically normal dogs subjected to inhalation anesthesia with isoflurane prior to euthanasia, 5 dogs with juvenile-onset dilated cardiomyopathy, and 5 dogs with adult-onset dilated cardiomyopathy). Procedures: Activity of mitochondrial complex I and complex IV was assayed spectrophotometrically in isolated mitochondria from left ventricular tissue obtained from the 4 groups of dogs. Results: Activity of complex I and complex IV was significantly decreased in anesthetized dogs, compared with activities in the control dogs and dogs with juvenile-onset or adult-onset dilated cardiomyopathy. Conclusions and Clinical Relevance: Inhalation anesthesia disrupted the electron transport chain in the dogs, which potentially led to an outburst of reactive oxygen species that caused mitochondrial dysfunction. Inhalation anesthesia depressed mitochondrial function in dogs, similar to results reported in other species. This effect is important to consider when anesthetizing animals with myocardial disease and suggested that antioxidant treatments may be beneficial in some animals. Additionally, this effect should be considered when designing studies in which mitochondrial enzyme activity will be measured. Additional studies that include a larger number of animals are warranted.