Nuclear Magnetic Resonance (NMR) Metabolomics: Current Applications in Equine Health Assessment.

Metabolomics can allow for the comprehensive identification of metabolites within biological systems, at given time points, in physiological and pathological conditions. In the last few years, metabolomic analysis has gained popularity both in human and in veterinary medicine, showing great potential for novel applications in clinical activity. The aim of applying metabolomics in clinical practice is understanding the mechanisms underlying pathological conditions and the influence of certain stimuli (i.e., drugs, nutrition, exercise) on body systems, in the attempt of identifying biomarkers that can help in the diagnosis of diseases. Proton Nuclear Magnetic Resonance spectroscopy (1H-NMR) is well tailored to be used as an analytical platform for metabolites' detection at the base of metabolomics studies, due to minimal sample preparation and high reproducibility. In this mini-review article, the scientific production of NMR metabolomic applications to equine medicine is examined. The research works are very different in methodology and difficult to compare. Studies are mainly focused on exercise, reproduction, and nutrition, other than respiratory and musculoskeletal diseases. The available information on this topic is still scant, but a greater collection of data could allow researchers to define new reliable markers to be used in clinical practice for diagnostic and therapeutical purposes.

Synovial Fluid Metabolome Can Differentiate between Healthy Joints and Joints Affected by Osteoarthritis in Horses.

Osteoarthritis (OA) is a common cause of lameness in sport horses with a significant economic impact. The prevention of OA is crucial since no effective treatment is available. This study aimed to apply untargeted metabolomic analysis to investigate the differences in synovial fluid (SF) composition between healthy and OA-affected joints in horses. SF collected from healthy (n.8) and OA (n.11) horses was analyzed using H-NMR analysis. Metabolomic analysis allowed 55 different metabolites to be identified and quantified in SF samples. Nineteen metabolites were found to be differently concentrated in OA compared to control horses. Synovial fluids from the OC group were found to be higher in 1,3-dihydroxyacetone but lower in tryptophan, phenylalanine, tyrosine, uridine, creatinine, creatine, glycine, choline, asparagine, glutamine, arginine, 3-hydroxybutyrate, valine, 2-hydroxyisovalerate, α-ketoisovaleric acid, 3-methyl-2-oxovalerate, 3-hydroxyisobutyrate, isoleucine, and methionine compared to the controls. A variety of SF metabolites significantly changed following joint disease, demonstrating the complex mechanism underlying osteoarthritis in horses and highlighting the value of applying the metabolomic approach in clinical research.

Donkey Colostrum and Milk: How Dietary Probiotics Can Affect Metabolomic Profile, Alkaline Sphingomyelinase and Alkaline Phosphatase Activity.

Positive results on animal health, feed efficiency, and milk's nutritional content have been obtained after oral administration of probiotics. The aim of the present study was therefore to evaluate the effect of dietary supplementation with high numbers of multispecies probiotic formulations on the milk metabolomic profiles of alkaline sphingomyelinase (alk-SMase) and alkaline phosphatase (ALP) in donkeys. Twenty animals were randomly allocated to receive either a normal diet (group B) or a supplemented diet (group A). Colostrum and milk samples were obtained within 48 h, at 15 days (supplementation start), and at 45 days after parturition. Different metabolomic profiles were observed between colostrum and milk, as were the concentrations of 12 metabolites that changed following 30 days of probiotic supplementation. Alk-SMase activity was found to be higher in donkey colostrum (vs. milk at 15 days); this enzyme, together with ALP, increased in milk after 30 days of probiotic supplementation. The results of the present study provide new insight into the complex changes in donkey colostrum and milk composition in the first 45 days of lactation and how the milk metabolome can be modulated by probiotic supplementation.

p62/SQSTM1 indirectly mediates remote multipotent mesenchymal cells and rescues bone loss and bone marrow integrity in ovariectomized rats.

Intramuscular administration of p62/SQSTM1 (sequestosome1)-encoding plasmid demonstrated an anticancer effect in rodent models and dogs as well as a high safety profile and the first evidence of clinical benefits in humans. Also, an anti-inflammatory effect of the plasmid was reported in several rodent disease models. Yet, the mechanisms of action for the p62 plasmid remain unknown. Here, we tested a hypothesis that the p62-plasmid can act through the modulation of bone marrow multipotent mesenchymal cells (MSCs). We demonstrated that a p62 plasmid can affect MSCs indirectly by stimulating p62-transfected cells to secrete an active ingredient(s) sensed by untransfected MSCs. When we transfected MSCs with the p62-plasmid, collected their supernatant, and added it to an untransfected MSCs culture, it switched the differentiation state and prompt osteogenic responses of the untransfected MSCs. According to an accepted viewpoint, ovariectomy leads to bone pathology via dysregulation of MSCs, and restoring the MSC homeostasis would restore ovariectomy-induced bone damage. To validate our in vitro observations in a clinically relevant in vivo model, we administered the p62 plasmid to ovariectomized rats. It partially reversed bone loss and notably reduced adipogenesis with concurrent reestablishing of the MSC subpopulation pool within the bone marrow. Overall, our study suggests that remote modulation of progenitor MSCs via administering a p62-encoding plasmid may constitute a mechanism for its previously reported effects and presents a feasible disease-preventing and/or therapeutic strategy.

Exploring the metabolome of seminal plasma in two different horse types: Light versus draft stallions.

The application of the 'omics' studies in the field of animal reproduction has been aimed at identifying novel biomarkers of fertility since the last few years. When assessing reproductive efficiency in horses, breed should also be taken into account as it can influence semen quality and fertility. Considering the growing interest in metabolomic analysis to evaluate male fertility, we aimed to investigate the metabolomic profile of seminal plasma in two different horse breeds. Twelve healthy stallions, n.6 American Quarter Horse (AQH) and n.6 Italian Draft Horse (IDH) stallions, regularly used for artificial insemination, were included in the study. Two semen collections, performed 30-day apart, were considered for the assessment of semen parameters including gel-free volume, spermatozoa (spz) concentration, spz progressive motility and seminal plasma analysis by 1 H-NMR.Semen characteristics differed between IDH and AQH (p < .05) as well as the first cycle conception rate that was higher in AQH than IDH (p = .001). Metabolomic analysis quantified 56 molecules in equine seminal plasma, with 11 metabolites showing different concentrations in IDH compared to AQH (p < .05).This study provided evidence of differences in seminal plasma metabolites' concentrations between studied horse types, highlighting specific metabolomic fingerprints characterizing AQH and IDH sperm.

Measuring Biochemical Variables and Serum Amyloid A (SAA) in Working Mules in Central Italy.

According to FAO reports, the global mule population counts about 9 million mules. This hybrid cross of a male donkey and a female horse is mainly used for draft purposes because they are thought to be strong and hardy animals. Most consider mules to be less susceptible to disease and fatigue compared to horses. Therefore, the aim of this study was to investigate the effects of fieldwork on biochemical variables and serum amyloid A in working mules. Blood samples were collected from 10 healthy, female, working mules before and after 8 h of fieldwork. According to statistical analysis, a significant influence (p < 0.05) of fieldwork was found on mules' electrolyte profile with increased levels of sodium, chloride, and calcium, as well as on blood urea nitrogen and creatinine. After a day of fieldwork, serum sodium, chloride, calcium, urea, and creatinine concentrations were increased, supporting decreases in body water and renal blood flow. However, without comparison to a group of mules that were not exercised yet maintained under similar ambient conditions, it is uncertain whether these changes can be attributed to exercise. Further, no change in SAA concentration was found after exercise, indicating that the work performed did not result in systemic inflammation.

Serum Amyloid A as a Promising Biomarker in Domestic Animals' Reproduction: Current Knowledge and Future Perspective.

The investigation of acute phase proteins in veterinary medicine has opened the doors towards the identification and use of new markers for a timely assessment of health status in both companion and food-producing animals. The aim of this paper is to review the literature available on the use of serum amyloid A (SAA), an acute phase protein, for the diagnosis and monitoring of reproductive disorders in animals. This review critically appraises the usefulness of such marker in clinical practice and summarizes the current state of knowledge. Recent advances in the diagnosis and monitoring of reproductive diseases are presented, highlighting where SAA evaluation may enhance early diagnostic tools for dogs, cats, cattle, and equines.

Assessment of serum amyloid A concentrations and biochemical profiles in lactating jennies and newborn Ragusano donkey foals around parturition and one month after foaling in Sicily.

A proper knowledge of biochemical parameters and inflammatory markers like serum amyloid A (SAA) is crucial in the monitoring of the first post-partum period in equids. Since no information is available on SAA for donkeys at this stage, 50 animals including jennies (n.10) and newborn foals (n.10) within 48 hr from foaling, and jennies (n.10) and foals (n.20) after 30 days from parturition were enrolled in the study to assess routine biochemical profile including SAA. Jennies showed higher alkaline phosphatase and lower bilirubins and cholesterol at 30 days of lactation compared to post-partum. Neonatal donkey foals showed significant higher concentrations of sodium, alkaline phosphatase, lactic dehydrogenase, blood urea nitrogen, creatinine and albumin within 48 hr of age, whilst higher values of phosphate and triglycerides were observed in older foals of 30 days of age. Significant higher SAA concentrations were recorded during the peripartum period in both jennies (25.95 ± 14.98 µg/ml) and newborn donkey foals (37.44 ± 19.75 µg/ml) compared to SAA values recorded in lactating jennies (2.38 ± 1.78 µg/ml) and in donkey foals (16.04 ± 18.14 µg/ml) at 30 days after parturition. The assessment of SAA in jennies and donkey foals around parturition and one month after foaling represents a valuable tool for the monitoring of health status during this stage when animals have to face with new challenges like the peak of lactation and extrauterine life adaptation respectively.

Equine grass sickness in italy: a case series study.

BACKGROUND: Equine grass sickness (EGS) has been reported in several European and extra-European countries. Despite this, no scientific paper about clinical cases of EGS in Italy has been published. EGS is a disease affecting almost exclusively horses kept on pasture, characterized by clinical signs related to lesions in autonomic nervous system (ANS), particularly in the enteric nervous system (ENS). According to clinical presentation, acute, subacute and chornic syndromes can be observed, with various sympthoms including dullness, anorexia, dysphagia, drooling of saliva, tachycardia, ptosis, patchy sweating and muscle fasciculations. In horses affected by acute forms, mild to moderate abdominal pain and large volumes of nasogastric reflux can be observed. The etiology is still speculative and many hypothesis have been proposed to explain the pathogenesis. CASE PRESENTATION: The present study describes four cases of EGS (one subacute and three chronic forms) occurred in Central Italy during early spring. In all the cases included in the study, the prognosis was poor and the horses were euthanized. The diagnosis was confirmed by histological examination of ANS or ENS. In two cases, in vivo diagnosis was obtained by histological examination of enteric bioptic samples collected during laparoscopy. CONCLUSIONS: EGS in Italy could be underdiagnosed and incidence understimated. Greater awareness should be applied in Italy for the inclusion of EGS in differential diagnosis for horses presenting clinical signs of abdominal pain associated or not with gastric reflux and muscular fasciculation. All the cases in this study concerned horses kept in the same pasture, confirming a possible premise-linked and management-linked factors on the ethiopathogenesis of EGS. The age of horses ranged from 2 to 6 years, that is consistent with the risk factor age for EGS (from 2 to 7 years of age). Previous suspected EGS diagnosis in the same livestock and recent cool dry weather were considered additional potential risk factors.

Ultrasonography Evaluation of Umbilical Structures in Clinically Healthy Donkey Foals during the First Week of Life.

The umbilicus is a potential access-point for pathogens in equine foal, causing umbilical infections and potentially life-threating illness. Early diagnosis based on ultrasonographic appearance and measurement is crucial to avoid severe complications and promptly implement appropriate therapy. This study ultrasonographically evaluates the umbilical remnants of donkey foals, in the first week of life. Fifteen healthy donkey foals were included in the study. The umbilical vein, arteries and urachus ultrasounds were performed at 24 h, 3 and 7 days of life, using a portable ultrasound machine and a 5-7.5 MHz multifrequency linear probe. The Kruskal-Wallis test and Dunn's multiple comparisons test were applied to verify differences in relation to time for all the umbilical remnants measured. Statistical significance was set at p < 0.05. No statistical differences were observed in relation to time regarding umbilical remnant measurements. A correlation was found between body weight and the left artery at T0. The regression of the umbilical remnant during the first week of life was slower compared with what was reported in equine foals but was comparable with the results on calves. Thus, the different regression timing needs be considered when evaluating donkey foals with umbilical remnant diseases within the first week of life.

Quantification of 17 Endogenous and Exogenous Steroidal Hormones in Equine and Bovine Blood for Doping Control with UHPLC-MS/MS.

A simple and fast analytical method able to simultaneously identify and quantify 17 endogenous and exogenous steroidal hormones was developed in bovine and equine blood using UHPLC-MS/MS. A total amount of 500 µL of sample was deproteinized with 500 µL of a mixture of methanol and zinc sulfate and evaporated. The mixture was reconstituted with 50 µL of a solution of 25% methanol and injected in the UHPLC-MS/MS triple quadrupole. The correlation coefficients of the calibration curves of the analyzed compounds were in the range of 0.9932-0.9999, and the limits of detection and quantification were in the range of 0.023-1.833 and 0.069-5.5 ppb, respectively. The developed method showed a high sensitivity and qualitative aspects allowing the detection and quantification of all steroids in equine and bovine blood. Moreover, the detection limit of testosterone (50 ppt) is half of the threshold admitted in plasma (100 ppt). Once validated, the method was used to quantify 17 steroid hormones in both bovine and equine blood samples. The primary endogenous compounds detected were corticosterone (range 0.28-0.60 ppb) and cortisol (range 0.44-10.00 ppb), followed by androstenedione, testosterone and 11-deoxycortisol.

Use of nutraceuticals in the stallion: Effects on semen quality and preservation.

Nutritional supplements are widely used in the equine industry with the aim of improving horse health, sports or reproductive performances. Over the years, a number of studies have focused on investigating the effects of several dietary compounds on the quality and preservation of stallion semen. This paper reviews the literature available on the use of nutritional supplementation for the improvement of reproductive performance and semen quality in equine species, critically appraising the benefits and negative effects of several compounds found in complementary feeds such as PUFAs from different sources, vitamins and antioxidants, carnitine and botanical extracts. Different nutraceuticals have been highlighted to improve stallion fertility by providing optimal levels of antioxidants, with the most promising results obtained by the combination of PUFAs and antioxidants that resulted to be essential for the maintenance of normal reproductive functions and the reduction of cryodamage in cooled and frozen equine semen.

The effects of 808-nm near-infrared laser light irradiation on actin cytoskeleton reorganization in bone marrow mesenchymal stem cells.

Tailoring the cell organelles and thus changing cell homeostatic behavior has permitted the discovery of fascinating metabolic features enabling enhanced viability, differentiation, or quenching inflammation. Recently, photobiomodulation (PBM) has been accredited as an effective cell manipulation technique with promising therapeutic potential. In this prospective, in vitro results revealed that 808-nm laser light emitted by a hand-piece with a flat-top profile at an irradiation set up of 60 J/cm2 (1 W, 1 W/cm2; 60 s, continuous wave) regulates bone marrow stromal cell (BMSC) differentiation toward osteogenesis. Considering the importance of actin cytoskeleton reorganization, which controls a range of cell metabolic activities, comprising shape change, proliferation and differentiation, the aim of the current work is to assess whether PBM therapy, using a flat-top hand-piece at higher-fluence irradiation on BMSCs, is able to switch photon signals into the stimulation of biochemical/differentiating pathways involving key activators that regulate de novo actin polymerization. Namely, for the first time, we unearthed the role of the flat-top hand-piece at higher-fluence irradiation on cytoskeletal characteristics of BMSCs. These novel findings meet the needs of novel therapeutically protocols provided by laser treatment and the manipulation of BMSCs as anti-inflammatory, osteo-inductive platforms.

Nutritional Supplements Containing Cardus mariano, Eucalyptus globulus, Gentiana lutea, Urtica urens, and Mallotus philippinensis Extracts Are Effective in Reducing Egg Shedding in Dairy Jennies (Equus asinus) Naturally Infected by Cyathostomins.

The increasing levels of anthelmintic resistance together with the restrictions in the use of drugs in food producing animals have enforced the search for sustainable alternative approaches for parasite control. The current study aimed to investigate the safety and the efficacy of a commercially available phytotherapic formulation against gastrointestinal strongyles in donkeys. Twenty-two Ragusana jennies (2.6 ± 0.5 years old) were assigned to two equal groups. One group was treated with two doses of a phytotherapic supplement Paraxitebio® containing Cardus mariano, Eucalyptus globulus, Gentiana lutea, Urtica urens, and Mallotus philippinensis, 14 days apart (Group A). One group was used as negative control (Group B). Individual fecal samples were collected at the beginning of the study (T-1), and after 7, 14, and 28 days (T7, T14, T28). Blood samples were collected on T-1 and T28 in order to assess changes in donkeys' hematological profile. After the initial rise in EPG values observed on T7, Group A showed a significant EPG decrease with lower eggs per gram (EPG) count compared to Group B on T28 and an overall fecal egg count reduction of 56.9% on the same time-point. Hematological parameters were within the normal physiological ranges for enrolled donkeys. However, significant differences in the values of RBCs, Hb, MCHC, MCV, WBCs, eosinophils, and basophils were recorded between groups after phytotherapic treatments, with Group A showing a general improvement in the hemogram picture. The phytotherapic supplement used in the current study was helpful in controlling intestinal parasites allowing a significant reduction in the fecal egg count 28 days after treatment. Further studies are needed to better explore the specific mode of action of the plant-derived formulation herein tested as well as to encourage their use as tool for the control of equine strongylosis under multimodal integrated approach in dairy donkey farms.

Exercise Induced Changes in Salivary and Serum Metabolome in Trained Standardbred, Assessed by 1H-NMR.

In the present study, data related to the metabolomics of saliva and serum in trained standardbred horses are provided for the first time. Metabolomic analysis allows to analyze all the metabolites within selected biofluids, providing a better understanding of biochemistry modifications related to exercise. On the basis of the current advances observed in metabolomic research on human athletes, we aimed to investigate the metabolites' profile of serum and saliva samples collected from healthy standardbred horses and the relationship with physical exercise. Twelve trained standardbred horses were sampled for blood and saliva before (T0) and immediately after (T1) standardized exercise. Metabolomic analysis of both samples was performed by 1H-NMR spectroscopy. Forty-six metabolites in serum and 62 metabolites in saliva were detected, including alcohols, amino acids, organic acids, carbohydrates and purine derivatives. Twenty-six and 14 metabolites resulted to be significantly changed between T0 and T1 in serum and saliva, respectively. The findings of 2-hydroxyisobutyrate and 3-hydroxybutyrate in serum and GABA in equine saliva, as well as their modifications following exercise, provide new insights about the physiology of exercise in athletic horses. Glycerol might represent a novel biomarker for fitness evaluation in sport horses.

Respiratory metabolites in bronchoalveolar lavage fluid (BALF) and exhaled breath condensate (EBC) can differentiate horses affected by severe equine asthma from healthy horses.

BACKGROUND: The use of an untargeted metabolomic approach to investigate biofluids of respiratory origin is of increasing interest in human and veterinary lung research. Considering the high incidence of equine asthma (> 14%) within horse population and the importance of this animal model for human disease, we aimed to investigate the metabolomic profile of bronchoalveolar lavage fluid (BALF) and exhaled breath condensate (EBC) in healthy and asthmatic horses. RESULTS: On the basis of clinical, endoscopic and BALF cytology findings, 6 horses with severe asthma (Group A) and 6 healthy horses (Group C) were included in the study. 1H-NMR analysis was used to identified metabolites in BALF and EBC samples. Metabolomic analysis allowed to identify and quantify 12 metabolites in BALF and seven metabolites in EBC. Among respiratory metabolites, myo-inositol, formate, glycerol and isopropanol in BALF, and methanol and ethanol in EBC, differed between groups (p < 0.05). CONCLUSIONS: The application of metabolomic studies to investigate equine asthma using minimally invasive diagnostic methods, such as EBC metabolomics, provided promising results. According to our research, the study of selective profiles of BALF and EBC metabolites might be useful for identifying molecules like myo-inositol and methanol as possible biomarkers for airways diseases in horses.

Anthelmintic Efficacy and Pharmacokinetics of Ivermectin Paste after Oral Administration in Mules Infected by Cyathostomins.

Ivermectin (IVM) is an anthelmintic compound commonly used off-label in mules due to its broad-spectrum of activity. Despite the general use of IVM in mules with the same dose and route of administration licensed for horses, significant pharmacokinetic differences might exist between horses and mules, as already observed for donkeys. The aim of the present study was to evaluate the pharmacokinetic profile and anthelmintic efficacy of an oral paste of IVM in mules naturally infected with cyathostomins. Fifteen adult mules with fecal egg counts (FEC) ≥200 eggs per gram (EPG), with exclusive presence of cyathostomins, were included in the study. All mules were orally treated with IVM according to the manufacturer's recommended horse dosage (200 µg/kg body weight). FECs were performed before (day-10 and day-3) and after treatment at days 14 and 28 by using a modified McMaster method. The FEC reduction (FECR%) was also calculated. Blood samples were collected from five animals at various times between 0.5 h up to 30 days post treatment to determine pharmacokinetic parameters. The maximum IVM serum concentration (Cmax) was 42.31 ± 10.20 ng/mL and was achieved at 16.80 ± 9.96 hours post-treatment (Tmax), area under the curve (AUC) was 135.56 ± 43.71 ng × day/mL. FECR% remained high (>95%) until the 28th day.