Changes in biomarkers of the redox status in whole blood and red blood cell lysates in canine hypothyroidism.

Hypothyroidism is the most commonly diagnosed endocrine disease in dogs. The objective of this study was to evaluate the changes in the redox status in canine hypothyroidism using whole blood (WB) and red blood cell (RBCs) lysates. For this purpose, a panel of five antioxidants and five oxidants biomarkers was measured in WB and RBCs lysates of 30 dogs with hypothyroidism, 26 dogs with non-thyroidal illnesses and 15 healthy dogs. The antioxidants measured were cupric reducing antioxidant capacity (CUPRAC), ferric reducing ability of plasma (FRAP), Trolox equivalent antioxidant capacity (TEAC), thiol and paraoxonase type-1 (PON-1). Oxidants measured include the total oxidant status (TOS), peroxide-activity (POX-Act), reactive oxygen-derived metabolites (d-ROMs), advanced oxidation protein products (AOPP) and thiobarbituric acid reactive substances (TBARS). WB showed a significant decrease of the antioxidants CUPRAC, TEAC and thiol, and also an increase in TBARS and a decrease in AOPP in dogs with hypothyroidism compared to healthy dogs. Meanwhile, RBCs lysates showed a significant increase in FRAP and PON-1 in dogs with hypothyroidism. The changes in the redox biomarkers in this study show that WB in canine hypothyroidism had a higher number of changes in biomarkers of the redox status than RBCs lysates, making it a promising sample type for the evaluation of the redox status in this disease. In addition, WB is easier and simpler to process than RBCs lysates and unlike serum, it does not have any hemolysis interference.

Validation of assays for measurement of oxidant compounds in saliva of pigs: Thiobarbituric acid reactive substances (TBARS), carbonyl, and reactive oxygen species (ROS).

Thiobarbituric acid reactive substances (TBARS), carbonyls and reactive oxygen species (ROS) are oxidant compounds that can provide useful information on the oxidative status. Pigs can be affected by oxidative stress in different situations including physiological conditions such as lactation and also in different diseases, and the measurement of these three analytes in saliva could be potentially useful as biomarkers of the redox status in this species. Assays for the measurement of TBARS and carbonyls by spectrophotometry and ROS by luminol-based chemiluminescence in pigs' saliva were analytically validated and were applied in saliva of pigs after an in vitro incubation with different doses of ascorbic acid (AA). All the assays showed a satisfactory analytical precision and accuracy. The 240 h-incubation of saliva samples with 60 mM of AA induced to an increased TBARS and carbonyls production. TBARS, carbonyls and ROS can be estimated in saliva of pigs by the assays validated in this report. In addition, these assays can detect changes in the concentration of these analytes associated to incubation of saliva samples with AA.

Changes in salivary analytes in cows due to the in vitro presence of feed.

BACKGROUND: The effect in a sialochemistry profile of the presence of usually available feed in dairy cows was evaluated by an in vitro experiment. For this purpose, a pooled clean saliva from five healthy dairy cows was incubated five times with a standard feed based on a total mixed ration (F), wheat hay (H), and grass (G). The salivary panel was integrated by biomarkers of stress (cortisol -sCor-, salivary alpha-amylase -sAA-, butyrylcholinesterase -BChE-, total esterase -TEA-, and lipase -Lip-), immunity (adenosine deaminase -ADA-), oxidative status (Trolox equivalent antioxidant capacity -TEAC-, the ferric reducing ability of saliva -FRAS-, the cupric reducing antioxidant capacity -CUPRAC-, uric acid, and advanced oxidation protein products -AOPP-), and enzymes, proteins, and minerals of general metabolism and markers of liver, muscle, and renal damage (aspartate aminotransferase -AST-, alanine aminotransferase -ALP-, γ-glutamyl transferase -gGT-, lactate dehydrogenase -LDH-, creatine kinase -CK-, creatinine, urea, triglycerides, glucose, lactate, total protein, phosphorus, and total calcium). RESULTS: Most of the evaluated analytes showed a coefficient of variations (CV) higher than 15% and/or significant changes compared with the clean saliva when feed was present. Some analytes, such as the oxidative status biomarkers (CV > 80%), AST (CV > 60%), or glucose (CV > 100%), showed significant changes with all the feed types tested. Others showed significant differences only with certain types of feed, such as LDH with F (CV > 60%) or triglycerides with F (CV > 100%) and H (CV > 95%). However, sCor or gGT remained unchanged (CV < 15%, P > 0.05) in all the treatments. CONCLUSIONS: The presence of feed can produce changes in most of the analytes measured in cows' saliva, being of high importance to consider this factor when saliva is used as a sample to avoid errors in the interpretation of the results.

Basics for the potential use of saliva to evaluate stress, inflammation, immune system, and redox homeostasis in pigs.

The use of saliva as a biological sample has many advantages, being especially relevant in pigs where the blood collection is highly stressful and painful, both for the animal and the staff in charge of the sampling. Currently one of the main uses of saliva is for diagnosis and detection of infectious diseases, but the saliva can also be used to measure biomarkers that can provide information of stress, inflammation, immune response and redox homeostasis. This review will be focused on the analytes that can be used for such evaluations. Emphasis will be given in providing data of practical use about their physiological basis, how they can be measured, and their interpretation. In addition, some general rules regarding sampling and saliva storage are provided and the concept of sialochemistry will be addressed. There is still a need for more data and knowledge for most of these biomarkers to optimize their use, application, and interpretation. However, this review provides updated data to illustrate that besides the detection of pathogens in saliva, additional interesting applicative information regarding pigs´ welfare and health can be obtained from this fluid. Information that can potentially be applied to other animal species as well as to humans.

Serum choline and butyrylcholinesterase changes in response to endotoxin in calves receiving intravenous choline administration.

Endotoxemia treatment options are still of interest due to high mortality and choline treatment is one of them because of its role in the cholinergic anti-inflammatory pathway. This study investigated serum choline and butyrylcholinesterase (BChE) responses, and their correlations with inflammatory, oxidative stress and tissue damage biomarkers, including paraoxanase-1 (PON1), and clinical signs in calves with endotoxemia and the effect of choline treatment in these responses. Healthy calves (n = 20) were divided equally into 4 groups: Control (0.9% NaCl, iv), Choline (C; 1 mg/kg/iv,once), Lipopolysaccharide (LPS; 2 µg/kg/iv,once) and LPS + C. Clinical and laboratory examinations were performed before and 0.5-48 h (hrs) after treatments. Following LPS administration, serum choline level increased at 0.5-24 h (P < .01), whereas serum BChE and PON1 level decreased at 48 h (P < .01) compared to their baselines. In LPS + C group, the increase in serum choline level was significantly higher (P < .01) than that of C and LPS groups. LPS did not decrease serum BChE levels significantly in calves treated with choline. Serum choline and BChE results correlated negatively with white blood cell count and positively (P < .001) with PON1 levels, oxidative stress index, inflammation and hepato-muscular injury markers. In conclusion serum choline and BChE may have a role in the pathophysiology of endotoxemia in calves. High serum choline concentration is associated with an improvement in response to LPS administration in calves treated with choline, probably by preventing the imbalances between oxidative stress and anti-oxidant capacity, preventing the serum BChE and PON1 decreases, and inhibition/attenuation of acute phase reaction and hepato-muscular injury in calves with endotoxemia.

Evaluation of adenosine deaminase in saliva and serum, and salivary α-amylase, in canine pyometra at diagnosis and after ovariohysterectomy.

An assay for adenosine deaminase (ADA) was validated in serum and saliva in dogs. Changes in ADA and salivary α-amylase activities were analysed in 26 bitches diagnosed with pyometra and compared with activities in 19 healthy bitches. All animals were classified according to the American Society of Anaesthesiologists (ASA) scoring for physical status. In the validation study, the ADA assay had an imprecision<12% and determination coefficients>0.90 in linearity under dilution experiments, with recoveries of 99.2-114.4%. On the day of presentation, salivary ADA activity was significantly higher in dogs with pyometra than in healthy dogs (median values 7.1IU/L vs. 0.8IU/L, respectively; P<0.01). ADA had a moderate positive correlation with leucocyte and band neutrophil counts, haptoglobin, salivary α-amylase and ASA score, and a low positive correlation with C-reactive protein. There were no significant differences in salivary α-amylase activity between dogs with pyometra and healthy dogs (57.3IU/L vs. 27.4IU/L, respectively). Salivary α-amylase had a low correlation with ASA grade, and leucocyte and band neutrophil counts. In 7/26 bitches with pyometra that were sampled 3 and 10days after ovariohysterectomy, there were no significant changes in α-amylase or ADA activities. These results indicate that ADA activity is increased in the saliva of bitches with pyometra, probably related to systemic inflammation.

Peroxidized mineral oil increases the oxidant status of culture media and inhibits in vitro porcine embryo development.

The use of oils with undetected alterations is a long-recognized problem for in vitro embryo production systems. Since peroxides in oils have been associated with reduced embryo production outcomes, our goals were (1) to evaluate the effects of a batch of mineral oil (MO) that was suspected to be altered on the in vitro production of pig embryos and (2) to determine oil peroxide values throughout culture and the transfer of oxidant agents from oil to culture media. Sunflower oil, which has a completely different chemical composition than MO but a higher oxidative status, and unaltered MO were used as controls. Oocyte maturation, fertilization and embryo development were affected differently depending on the oil overlay used. While the suspected MO was not able to sustain in vitro maturation and fertilization, the oocytes incubated in the presence of sunflower oil were matured and fertilized similarly to those of the unaltered MO group. Moreover, the cleavage rate of presumed zygotes cultured under the suspected MO was severely reduced compared with those cultured under the other oils, and none of the cleaved embryos developed to the blastocyst stage. Although the cleavage rates in the sunflower oil and unaltered MO groups were similar, embryos cultured under sunflower oil also failed to develop to the blastocyst stage. Our results revealed that the suspected MO and sunflower oil had similar levels of peroxides and that these levels were much higher than those of the unaltered MO. The total oxidant status was higher in media incubated under peroxidized oils than in fresh media or media incubated without an oil overlay or under unaltered MO, indicating that oxidant agents were transferred to the incubation media. However, unlike the sunflower oil group, the culture media incubated under the suspected MO had high levels of total oxidant status and low levels of hydrogen peroxide and reactive oxygen species, suggesting the presence of other unknown oxidant agents in that oil. These results indicate that a peroxidized MO overlay dramatically decreases embryo production outcomes. This decrease could be associated with the higher peroxide values of the oil but cannot be explained by the levels of hydrogen peroxide and reactive oxygen species transferred from the oil to the culture media. It is likely that different oxidant agent(s) and/or other toxic compounds present in the peroxidized MO are responsible for its damaging effects on oocytes and embryos.

Serum biomarkers of oxidative stress in dogs with idiopathic inflammatory bowel disease.

The objective of this work was to study and compare a panel of various serum biomarkers evaluating both the antioxidant response and oxidative damage in dogs with idiopathic inflammatory bowel disease (IBD). Eighteen dogs with IBD and 20 healthy dogs were enrolled in the study. Trolox equivalent antioxidant capacity (TEAC), cupric reducing antioxidant capacity (CUPRAC), ferric reducing ability of the plasma (FRAP), total thiol concentrations, and paraoxonase 1 (PON1) activity were evaluated in serum to determine antioxidant response. To evaluate oxidative status, ferrous oxidation-xylenol orange (FOX), thiobarbituric acid reactive substances (TBARS) and reactive oxygen species production (ROS) concentrations in serum were determined. Mean concentrations of all antioxidant biomarkers analyzed, with exception of FRAP, were significantly lower (P < 0.0001) in the sera of dogs with IBD than in healthy dogs. The oxidant markers studied were significantly higher (P < 0.0001) in sera of dogs with IBD than in healthy dogs. These findings support the hypothesis that oxidative stress could play an important role in the pathogenesis of canine IBD.