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This study aims to evaluate the circadian and circannual variations in a panel of analytes in horse saliva that have been previously described as biomarkers related to stress and disease, in order to interpret them correctly when they are measured in this species. This panel of analytes integrated cortisol, salivary alpha-amylase (sAA), lipase (Lip), total esterase (TEA), butyrylcholinesterase (BChE), adenosine deaminase (ADA), γ-glutamyl transferase (gGT), creatine kinase (CK), urea, total bilirubin, total protein (TP), and phosphorus. These analytes were measured in saliva obtained from a population of five clinically healthy mares from 06:30 to 20:30, every 2 h over two consecutive days in two different photoperiod seasons, winter and spring. The temperature and relative humidity did not change between the two consecutive days sampled in each sampled season, and no thermal discomfort was observed. Changes throughout the course of the day were observed for cortisol, sAA, TEA, BChE, ADA, and CK. However, a circadian pattern was only observed for cortisol, TEA, BChE, ADA, and CK. Moreover, the values obtained for sAA, Lip, and BChE were significantly different between seasons, with different daily rhythms for cortisol, TEA, BChE, and ADA depending on the season. In conclusion, this pilot study indicates that the time of the day and the season influence salivary analytes in horses, showing a rhythmic pattern for cortisol, TEA, BChE, ADA, and CK. These factors should thus be taken into consideration for the interpretation of analytes in horse saliva.

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Oxidative stress can affect animal's health and the quality of its final products. The oxidative status can be evaluated by the measurement of both oxidant and antioxidant biomarkers. The use of saliva as a sample is preferable to blood, as individuals with limited training can collect it easily and non-invasively with minimal stress to the animal. The aim of this study was to perform an analytical validation of automated assays of the ferric reducing ability of plasma (FRAP), the cupric reducing antioxidant capacity (CUPRAC), the Trolox equivalent antioxidant capacity (TEAC) and uric acid as antioxidant biomarkers and of the advanced oxidation protein products (AOPP) and hydrogen peroxide (H2O2) as oxidant biomarkers in saliva samples of sheep, and to evaluate their possible changes after stress induced by shearing. All assays produced acceptable results in the analytical validation, from which it can be concluded that oxidative stress biomarkers such as FRAP, CUPRAC, TEAC, uric acid and AOPP and H2O2 can be measured in sheep saliva. In addition, acute stress due to shearing could produce an oxidative stress response in sheep and subsequently increase antioxidants in order to protect cells from damage.

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The objective of this study was to develop a time-resolved immunofluorometric assay (TR-IFMA) for quantification of salivary alpha-amylase in sheep. For that purpose, after the design of the assay, an analytical and a clinical validation were carried out. The analytical validation of the assay showed intra- and inter-assay coefficients of variation (CVs) of 6.1% and 10.57%, respectively and an analytical limit of detection of 0.09 ng/mL. The assay also demonstrated a high level of accuracy, as determined by linearity under dilution. For clinical validation, a model of acute stress testing was conducted to determine whether expected significant changes in alpha-amylase were picked up in the newly developed assay. In that model, 11 sheep were immobilized and confronted with a sheepdog to induce stress. Saliva samples were obtained before stress induction and 15, 30, and 60 min afterwards. Salivary cortisol was measured as a reference of stress level. The results of TR-IFMA showed a significant increase (P < 0.01) in the concentration of alpha-amylase in saliva after stress induction. The assay developed in this study could be used to measure salivary alpha-amylase in the saliva of sheep and this enzyme could be a possible noninvasive biomarker of stress in sheep.

L'objectif de la présente étude était de développer un test immunofluorométrique en temps résolu (TIMF-TR) pour la quantification de l'alpha-amylase salivaire chez le mouton. À cette fin, suite au design du test, une validation analytique et clinique fut effectuée. La validation analytique du test a montré des coefficients de variation (CV) intra- et inter-tests de 6,1 % et 10,57 %, respectivement, et une limite de détection analytique de 0,09 ng/mL. Le test a également montré un haut niveau de précision, tel que déterminé par la linéarité suite aux dilutions. Pour la validation clinique, un modèle de test de stress aigu a été mené afin de déterminer si des changements significatifs attendus de l'alpha-amylase étaient détectés dans le nouveau test développé. Dans ce modèle, 11 moutons étaient immobilisés et confrontés avec un chien de berger afin d'induire le stress. Des échantillons de salive ont été obtenus avant l'induction du stress et 15, 30, et 60 min par la suite. Le cortisol salivaire a été mesuré à titre d'indicateur de référence du stress. Les résultats du TIMF-TR ont montré une augmentation significative (P < 0,01) de la concentration d'alpha-amylase dans la salive après l'induction du stress. Le test développé au cours de cette étude pourrait être utilisé afin de mesurer l'alpha-amylase salivaire dans la salive de mouton et cet enzyme pourrait être un biomarqueur non-invasif du stress chez le mouton.(Traduit par Docteur Serge Messier).

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El objetivo de este estudio fue evaluar la producción y calidad forrajera de variedades locales de algarroba(Vicia monanthos), almorta (Lathyrus sativus), guisante forrajero (Pisum sativum), veza común (Vicia sativa),alverjón (Vicia narbonensis) y yero (Vicia ervilia). El estudio se realizó en tres localidades de la provincia deAlbacete, España, durante tres años consecutivos de cultivo. Los muestreos se realizaron en abril, junio y julioy en cada muestra se determinaron los parámetros cualitativos, materia seca (% MS), proteína bruta (% PB),fi bra ácido detergente (% FDA), fi bra neutro detergente (% FDN) y energía metabolizable (Mcal) y a partir deellos la producción (kg MS/ha). En el muestreo de abril la especie de mayor rendimiento (P ≤ 0,05) fue el yero(1.127 kg MS/ha, 20,98% PB y 2,327 Mcal/kg MS) seguido de veza común (924 kg MS/ha, 23,01% PB 2,498Mcal/kg MS). En el muestro de junio las especies de mayor rendimiento (P ≤ 0,05) fueron vicia común (1.895kg MS/ha, 20,01% PB y 2,247 Mcal/kg MS) y el yero (1.802 kg MS/ha, 16,12% PB y 2,033 Mcal/kg MS). Enjulio también fueron el yero (2.880 kg MS/ha, 13.98 % PB y 1,906 Mcal/kg MS) y la veza común (2.777 kgMS/ha, PB 14,97% y 2,043 Mcal/kg MS) las especies de mayor rendimiento (P ≤ 0.05). Nuestros resultadosindican que en las condiciones de del estudio el yero y la veza común fueron las especies de más productivas ypor tanto de mayor interés como forraje, desde el fi nal del período vegetativo hasta la plena madurez(AU)

The aim of this study was to evaluate the forage yield and quality of local varieties of Bard vetch (Viciamonanthos), Grass pea (Lathyrus sativus), Field pea (Pisum sativum), Common vetch (Vicia sativa), Narbonvetch (Vicia narbonensis) and Bitter vetch (Vicia ervilia). The trial was carried out in three locations over three consecutive years of harvest. Quantitative (kg DM/ha) and qualitative variables (dry mater (%DM), crudeprotein (%PB), acid detergent fi ber (%FAD), neutral detergent fi ber (%FND) and metabolizable energy (Mcal)were determined for April, June and July. Highest yield in the April sampling was Bitter vetch (1.127 kg DM/ha, 20,98 %PB and 2,327 Mcal/kg DM), followed by Common vetch (924 kg DM/ha, 23,01 %PB 2,498 Mcal/kg DM). In the June, the highest yield species were Common vetch (1.895 kg DM/ha, 20,01 %PB and 2,247Mcal/kg DM) and Bitter vetch (1.802 kg DM/ha, 16,12 %PB and 2,033 Mcal/kg DM). For July, the highestyield was for Bitter vetch (2.880 kg DM/ha, 13,98 %PB and 1.906 Mcal/kg DM), followed by Common vetch(2,777 kg DM/ha, 14.97 %PB and 2,043 Mcal/kg DM). Our results sown that, under the conditions of our study,Common vetch and Bitter vetch were the spices of greater interest as fodder for ruminants from the end of thevegetative period to the crop attained full ripeness(AU)

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