Changes in the salivary metabolome in canine hypothyroidism: A pilot study.

Hypothyroidism is the most commonly diagnosed endocrine disorder in dogs. It produces a deficiency of thyroid hormones which impacts negatively the dog's quality of life. The objective of this study is to evaluate the possible changes in the salivary metabolic profile in dogs with hypothyroidism. For this purpose, targeted metabolomics analysis performed by LC/MS analysis was made in saliva samples from a group of dogs with hypothyroidism and a group of healthy dogs. Twenty-three metabolites showed a significant decrease between hypothyroid and healthy dogs, most of these associated with thyroid hormone synthesis, catecholamine synthesis, and tyrosine and phenylalanine metabolism. Based on the results, it can be stated that hypothyroidism produces changes in the metabolome of saliva and some of them can reflect the metabolic changes presented in the disease and could serve as a potential biomarker of this condition.

Seminal Plasma Antioxidants Are Related to Sperm Cryotolerance in the Horse.

The objective of this study was to determine the relationship of enzymatic (superoxide dismutase, SOD; glutathione peroxidase, GPX; catalase, CAT; and paraoxonase type 1, PON1) and non-enzymatic antioxidants (measured in terms of: Trolox equivalent antioxidant capacity, TEAC; cupric-reducing antioxidant capacity, CUPRAC; and ferric-reducing ability of plasma, FRAP), as well as the oxidative stress index (OSI) in seminal plasma (SP) with the resilience of horse sperm to freeze-thawing. Twenty-one ejaculates (one per individual) were collected and split into two aliquots: the first was used to harvest the SP and assess the activity levels of antioxidants and the OSI, and the second one was cryopreserved. The following post-thaw sperm quality parameters were evaluated: sperm motility, plasma membrane and acrosome integrity, mitochondrial membrane potential, intracellular levels of reactive oxygen species (ROS), and plasma membrane lipid disorder. Based on post-thaw total motility (TM) and plasma membrane integrity (SYBR14+/PI−), ejaculates were hierarchically (p < 0.001) clustered into two groups of good (GFE) and poor (PFE) freezability. The SP activity levels of PON1, SOD, and TEAC were higher (p < 0.05) in GFE than in PFE, showing a positive relationship (p < 0.05) with some sperm motility parameters and with plasma membrane (PON1 and TEAC) and acrosome (SOD and TEAC) integrity. In contrast, OSI was higher (p < 0.05) in the SP of PFE than in that of GFE, and was negatively correlated (p < 0.05) to some sperm motility parameters and to plasma membrane and acrosome integrity, and positively (p < 0.05) to the percentage of viable sperm with high plasma membrane lipid disorder. In conclusion, enzymatic (PON1 and SOD) and non-enzymatic (TEAC) antioxidants of SP are related to horse sperm cryotolerance. In addition, our results suggest that PON1 could be one of the main antioxidant enzymes involved in the control of ROS in this species. Further investigation is needed to confirm the potential use of these SP-antioxidants and OSI to predict sperm cryotolerance in horses.

Changes in Biomarkers of Redox Status in Saliva of Pigs after an Experimental Sepsis Induction.

Saliva from pigs is gaining attention as an easy sample to obtain, being a source of biomarkers that can provide information on animal health and welfare. This study aimed to evaluate the changes that can occur in salivary biomarkers of the redox status of pigs with an experimentally induced sepsis. For that, the cupric reducing antioxidant capacity (CUPRAC), ferric reducing ability of saliva (FRAS), Trolox equivalent antioxidant capacity (TEAC), advanced oxidation protein products (AOPP), ferrous oxidation-xylenol orange (FOX), peroxide activity (POX-Act), and reactive oxygen-derived compounds (d-ROMs) were measured in the saliva of pigs with experimentally induced sepsis by endotoxin lipopolysaccharide (LPS), non-septic inflammation induced by turpentine, and in healthy individuals before and after 3 h, 6 h, 24 h, and 48 h. AOPP, POX-Act, and d-ROMs in the sepsis group were higher than in the control from 3 h to 24 h after the inoculation. CUPRAC, FRAS, and TEAC were higher in sepsis than the control group at 24 h. These changes were of higher magnitude than those that occurred in the turpentine group. In conclusion, our findings reveal that sepsis produces changes in salivary biomarkers of redox status, which opens the possibility of using them as potential biomarkers in this species.

Changes in Oxidative Status Biomarkers in Saliva and Serum in the Equine Gastric Ulcer Syndrome and Colic of Intestinal Aetiology: A Pilot Study.

Changes in the oxidative status of the blood of horses suffering from gastric ulcers and colic of intestinal aetiology (CIE) have been reported. However, saliva can also be a source of biomarkers of oxidative status. Therefore, this study aims to validate automated assays for the measurement of oxidative status biomarkers (ferric reducing ability of saliva/serum-FRAS/FRAP, cupric reducing antioxidant capacity-CUPRAC, the Trolox equivalent antioxidant capacity-TEAC, uric acid, and advanced oxidation protein products-AOPP) in the saliva and serum of horses, to assess their changes in the different ulcer gastric diseases (squamous-ESGD and glandular-EGGD) and CIE, and to evaluate their relationship with serum amyloid A (SAA), adenosine deaminase (ADA), and the systemic inflammatory response syndrome (SIRS) status. The assays showed a low imprecision and good linearity with enough sensitivity in both fluids. In EGGD, higher levels of FRAS, uric acid, and AOPP in saliva were observed compared to the healthy group, correlating with the salivary ADA levels. Horses with CIE showed increases in uric acid concentrations in serum associated with their SIRS status and outcome of the disease. In conclusion, analytes related to the oxidative status can be measured in the saliva and serum from horses by automated assays, and some of them can potentially be assessed as biomarkers in horses with gastric ulcers and CIE.

Impact of Seminal Plasma Antioxidants on Donkey Sperm Cryotolerance.

This study investigated whether the activities of the antioxidant components of donkey seminal plasma (SP)-both enzymatic (superoxide dismutase (SOD), catalase-like (CAT), glutathione peroxidase-like (GPX), and paraoxonase type 1 (PON1)) and non-enzymatic (measured in terms of total thiol, copper-reducing antioxidant capacity (CUPRAC), ferric-reducing ability of plasma (FRAP), and Trolox equivalent antioxidant capacity (TEAC))-and oxidative stress index (OSI) are related to sperm cryotolerance. For this purpose, 15 ejaculates from jackasses (one per individual) were collected and split into two aliquots. The first one was used for measuring the activities levels of enzymatic and non-enzymatic antioxidants and OSI in SP, whereas the other aliquot was cryopreserved. Before cryopreservation, sperm quality parameters (concentration, motility, and viability) were evaluated. After thawing, sperm motility, plasma membrane integrity, lipid disorder, mitochondrial membrane potential, reactive oxygen species (ROS), and calcium intracellular levels were also determined. Based on the percentages of total motility (TM) and of sperm with an intact plasma membrane (SYBR14+/PI-) after thawing, samples were classified as good-freezability (GFE) or poor-freezability (PFE) ejaculates through cluster analyses. The SP activity levels of enzymatic (SOD and PON1) and non-enzymatic antioxidants (CUPRAC, FRAP, and TEAC) were higher (p < 0.05) in GFE than in PFE, whereas SP-OSI was higher (p < 0.05) in PFE than in GFE. In addition, the activity levels of SOD, PON1, GPX, CUPRAC, FRAP, and TEAC were positively (p < 0.05) related to post-thaw sperm motility and plasma membrane integrity and negatively to intracellular ROS levels. The SP-OSI was negatively correlated (p < 0.05) to post-thaw sperm quality parameters and positively to intracellular ROS levels. It can thus be concluded that donkey SP antioxidants are related to sperm cryotolerance and that measurements of antioxidants PON1, SOD, CUPRAC, FRAP, and TEAC, as well as SP-OSI, could be used as markers of sperm cryotolerance. Further research addressing the relationship of these antioxidants and SP-OSI with sperm cryotolerance and their potential use as freezing markers is warranted.

Proteomics in dogs: a systematic review.

Proteomic analysis is having a rapid development as a method for the detection of biomarkers of diseases in dogs. Dogs in addition to their importance as companion animals, serve as important animal models for research. This study aims to systematically review evidence regarding the studies performed in proteomics in dogs, and specifically those made in serum, saliva, urine and/or plasma. Information searched in October 2020, January 2021 and August 2021, for English language publications of the last decade (2010-2020) were obtained from electronic databases. Screening, data extraction and risk of bias assessment were undertaken by two investigators. The risk of bias was evaluated using the Review Manager (RevMan 5) tool. Meta-analysis and case report studies were not included in this review. Through the screening process a total of 557 publications were identified after the removal of duplicates. Out of these, 65 were fully evaluated and 44 of these were included in the review. Most studies evaluated the proteome of disease and compared it with a healthy population, and most of the articles included were made on serum, followed by saliva. The overall risk of bias for all studies was high, due to an absence in the generation of random sequence. Overall proteomic analysis has allowed the discovery of new physiopathological pathways of diseases and potential biomarkers in the dog, which are addressed in this review.

Untargeted metabolomic profiling of serum in dogs with hypothyroidism.

Hypothyroidism is one of the most commonly diagnosed endocrine disease in dogs. The clinical signs are caused by a deficiency of the active thyroid hormones triiodothyronine (T3) and thyroxine (T4) and have a negative impact on dog's quality of life. We hypothesized that serum metabolic profile varies between healthy dogs and dogs with hypothyroidism. Twenty serum samples from dogs with hypothyroidism and 20 from healthy dogs were used for untargeted metabolomics analysis performed by LC/MS analysis. Fifteen metabolites showed significant changes between hypothyroid and healthy dogs, being the pentose phosphate pathway (PPP), aminoacyl-tRNA biosynthesis and pyrimidine metabolism the principal pathways altered in hypothyroidism. Specifically, metabolites such as D-gluconic acid and L-Isoleucine may potentially act as biomarkers of disease.

Characterization of total adenosine deaminase activity (ADA) and its isoenzymes in saliva and serum in health and inflammatory conditions in four different species: an analytical and clinical validation pilot study.

BACKGROUND: Measurement of adenosine deaminase (ADA) can provide information about cell-mediated immunity. This report's objective was to study the enzymatic activity of total ADA (tADA) and its isoenzymes ADA1 and ADA2 in canine, equine, porcine, and bovine serum and saliva and their changes in different inflammatory situations in each species. Besides, an automated method for ADA2 measurement was developed and validated. RESULTS: tADA was present in serum and saliva of healthy animals of the four species. Erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) concentration of 0.47 mM was needed for ADA1 inhibition in canine and porcine samples (serum and saliva) and bovine saliva, whereas for equine saliva 0.94 mM was needed. ADA2 activity was not detected in bovine serum and was very low or absent in equine serum and bovine saliva. An automated procedure to measure ADA2 consisting of adding EHNA to a commercial reagent for tADA measurement provided repetitive (coefficients of variation < 8.8% in serum and < 10% in saliva) and accurate (linearity of serial sample dilutions with R2 > 0.90) results, being equivalent to a manual incubation of the sample with EHNA at a similar concentration. Salivary tADA, as well as ADA1 and ADA2, were higher in dogs with leishmaniosis, horses with acute abdominal disease and pigs with lameness than in healthy animals. tADA and isoenzymes in saliva showed a positive significant correlation with serum ferritin in dogs (r = 0.602, P < 0.01; r = 0.555, P < 0.05; and r = 0.632, P < 0.01; respectively for tADA, ADA1 and ADA2) and serum C-reactive protein in pigs (r = 0.700, P < 0.01, for both tADA and ADA1; r = 0.770, P < 0.001, for ADA2), whereas salivary ADA2 significantly correlated with serum amyloid A in horses (r = 0.649, P < 0.01). In cows, salivary tADA and ADA1 significantly increased after calving, correlating with total white blood cell count (r = 0.487, P < 0.05, for both tADA and ADA1). CONCLUSIONS: The activity of total ADA and its different isoenzymes, can be measured in serum and saliva of dogs, horses, pigs and cows by a simple and fast procedure described in this report. When measured in saliva, these analytes correlated with other biomarkers of inflammation and it could potentially be used as a biomarkers of inflammation and immune activation in the species of this study.