Fecal thyroid hormone metabolites in wild ungulates: a mini-review.

This review aims to analyse the fluctuations of fecal thyroid hormone metabolites (FTMs) related to environmental and individual variables in different species of wild ungulates and provide a collection of assay methods. The great advantage of fecal sampling is being completely non-invasive. A systemic search was conducted from 2019 to 2024, using data sources PubMed, Scopus, Web of Science, and the World Wide Web, and ten studies were found on this topic. Three studies used the radioimmunoassay method for FTMs analysis, while the others used a less expensive enzyme-linked immunosorbent assay. Most of these papers validated the method for the species-specific matrix. Related to the studied variables, some authors analysed FTM fluctuations only concerning individual variables, and others in response to both. Temperature and fecal cortisol metabolites (FCMs) were the most studied environmental and individual variables, respectively. Since FTMs are an integrative measure of plasma thyroid hormones, the information obtained from a non-invasive-assay method regarding wild ungulate physiology is becoming of great interest to the scientific community.

An Overview on Assay Methods to Quantify ROS and Enzymatic Antioxidants in Erythrocytes and Spermatozoa of Small Domestic Ruminants.

The present review aims to provide an overview of the assay methods for the quantification of ROS and principal enzymatic antioxidants as biomarkers of oxidative stress in erythrocytes and spermatozoa of small domestic ruminants. A complete literature search was carried out in PubMed, Scopus and the World Wide Web using relevant keywords and focusing on the last five years (2018-2023). Among spectrophotometry, fluorometry and chemiluminescence, the most widely used method for ROS assay is fluorometry, probably because it allows to simultaneously assay several ROS, using different probes, with greater economic advantages. Regarding intracellular antioxidant enzymes, recent literature reports only spectrophotometric methods, many of which use commercial kits. The use of a less sensitive but cheapest method is suitable because both erythrocytes and spermatozoa samples are highly concentrated in domestic ruminant species. All methods considered in this review have been found to be appropriate; in general, the differences are related to their costs and sensitivity. Quantification of ROS and enzymatic antioxidant activity in erythrocytes and spermatozoa may find application in the study of the welfare and health status of small domestic ruminants for monitoring livestock production.

Application of the "echo-synch protocol" to advance pregnancy onset in ewe lambs at the first reproductive season.

Introduction: This study assessed the efficacy and economic impact of a reproductive protocol based on repeated ultrasound scanning (US) associated with the use of GnRH to advance pregnancy onset in ewe lambs. Methods: Prepubertal ewe lambs (n = 133) were divided into three weight groups (High: HW n = 35; Medium: MW n = 65; Low: LW n = 33). Thereafter, animals were randomly allocated into two subgroups: GnRH, ewe lambs treated with GnRH analog and then exposed to rams; CTR, ewe lambs exposed to rams only. CTR groups were joined with rams as a single flock. GnRH groups were kept separate from rams receiving a single dose of gonadorelin (40 µg/head) and then were evaluated after a week of US. Animals showing corpora lutea received an injection of PGF2α analog (100 µg/head) and then were joined with rams. The remaining ewe lambs received a second dose of gonadorelin and were kept separate from the rams. After another week, animals were checked again and the ones showing corpora lutea were injected with the PGF2α analog, while the others received a third injection of gonadorelin. On the same day, all the animals were joined with rams. Pregnancies were confirmed within 30 days by US. The efficacy of the protocol was determined by assessing differences in the number of days required to achieve pregnancy rates of 25, 50, and 75% and in the total costs and incomes from birth to the end of first lactation within the groups. Results: The GnRH-MW group showed the best performances in reaching the threshold pregnancy rates of 25, 50, and 75%, but the effect of treatment was significant only at the 25% threshold (p < 0.01). Both low groups displayed an overall poorer performance at 50 and 75% thresholds than medium and high-weight groups (p = 0.01 and p < 0.01, respectively). The GnRH administration did not advance pregnancy onset in GnRH-HW compared with CTR-HW. In the balance between costs and income, the HW-CTR and MW-GnRH groups showed higher gross margins than the other groups. Conclusion: Using the US/GnRH protocol in ewe lambs appears technically and economically effective in animals that have not reached the optimal weight at the first breeding season, advancing ewe lambs' pregnncies and increasing farm profitability.

Measurement of fecal T3 metabolite levels in sheep: Analytical and biological validation of the method.

Introduction: Biological sample collection from wild and farms animals is often associated with difficulties related to the handling and restraint procedures, and most of the time it could induce stress, altering the welfare and physiological homeostasis. The analysis of fecal T3 metabolites (FTMs) allows to test samples collected in a non-invasive manner, providing several information about the animal's physiological conditions and the effects related to environmental and nutritional variations. This procedure has found wide application in wild species, but less in domestic ones. Methods: The aim of this work was to validate the use of an immuno-enzymatic competitive ELISA kit, designed for T3 quantification in human blood serum samples, for the assessment of FTMs in the sheep. For the analytical validation, precision, recovery and parallelism were evaluated; for biological validation the variations of FTMs in relation to age, sex and the physiological status of the animal were determined. Results: After a verification of the precision (RSD % < 15%), mean recovery (75%) and parallelism (CV% < 10%), the kit was used to measure FTMs in cyclic, pregnant, and early lactating ewes as well as in rams and ewe lambs. The results showed that FTMs concentrations in pregnant ewes were significantly lower (p < 0.05) than in cyclic and early lactation ones. Furthermore, there were no significant differences in FTMs levels between ewes and rams, while in lambs FTMs levels were higher than in adults (p < 0.001). Conclusion: In conclusion the present study demonstrates that FTMs can be reliably and accurately determined in sheep feces, using an ELISA kit formulated for human serum T3 assay. The application of this method in the livestock sector could allow to improve our knowledge about the response of animals to different physiological and environmental conditions, and thus assess their welfare.

Non-invasive Assay for Measurement of Fecal Triiodothyronine (T3) Metabolite Levels in European Mouflon (Ovis aries musimon).

Thyroid hormones (THs) are important indicators of metabolism and animal health. Traditionally, they have been determined from blood or urine samples. However, as their collection may be stressful and requires ethical approval, alternative non-invasive matrices are preferred when dealing with wild animals. Triiodothyronine (T3) is the active form of THs in blood and the major metabolite excreted in feces. This creates the ideal conditions for its assay in fecal samples. Fecal sampling eliminates the stress of the animals and the need to physically capture them. However, in wild species it is rare to find species-specific kits for the hormone assay. So, the objective of this work was to validate a method for the quantification of T3 metabolite (FTM) levels in feces of European mouflon by using an economic and easily available ELISA kit designed to quantify T3 in human plasma. Analytical and biological validations were performed in feces collected from 10 mouflons (5 ewes and 5 rams). An efficient liquid-extraction method was optimized. Precision, dilution linearity, parallelism, recovery and stability of T3 in fecal samples were calculated. Obtained data were considered acceptable according to international guidelines. The reliability of the results was verified comparing human plasma and mouflon fecal samples fortified with the same T3 standard solutions. The biological validation showed higher FTM levels in March compared to June, and no differences between mouflon ewes and rams. The validation of the present method provides a non-invasive and affordable tool for the quantification of FTM in European mouflon.