Metabolomic Profiles in Starved Light Breed Horses during the Refeeding Process.

The large population of emaciated horses continues to be an issue troubling the equine industry. However, little is known regarding the collection of equine metabolites (metabolome) during a malnourished state and the changes that occur throughout nutritional rehabilitation. In this study, ten emaciated horses underwent a refeeding process, during which blood samples were collected for a blood chemistry panel and metabolomics analysis via ultrahigh performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS). Significant differences among blood chemistry analytes and metabolite abundance during the critical care period (CCP; Days 1-10 of rehabilitation) and the recovery period (RP; the remainder of the rehabilitation process) were observed. Potentially toxic compounds, analytes related to liver, kidney, and muscle function, as well as energy-related metabolites were altered during the refeeding process. The combination of blood chemistry and metabolomics analyses on starved equine during rehabilitation provide vital biological insight and evidence that the refeeding process has a significant impact on the equine metabolome.

Influence of Reproductive Status on Equine Serum Proteome: Preliminary Results.

The reproductive cycle and early pregnancy represent dynamic physiological states in mammals, but mechanisms involved in early pregnancy in the domestic horse remain poorly understood. Proteins in uterine secretions have been studied, but the proteome of peripheral serum during various reproductive states has not been investigated. This study characterized and compared the serum proteome in the domestic horse during various reproductive states. Serum was collected from three mares during: (1) estrus (day [d] -1; d 0 = ovulation), (2) diestrus (d 12.5, non-mated), (3) early pregnancy (d 12.5, pregnant), and (4) nonpregnant (d 12.5, unsuccessfully mated) states. Serum proteins in each sample were analyzed by Nano LC-MS/MS, and 308 proteins were identified. Differentially-expressed proteins (DEP; > 1.5-fold or < - 0.5-fold) were identified by comparison of protein relative abundance between reproductive states: (1) diestrus compared to estrus (DEP = 71), (2) pregnant compared to diestrus (DEP = 72), and (3) non-pregnant compared to pregnant (DEP = 81). DEPs were analyzed for biological function using PANTHER (pantherdb.org). Several pregnancy-specific proteins previously identified in equine pregnant histotroph, including Apolipoprotein A-I, Complement C3, and Histone H4, were detectable in the serum. The ability to detect these biomarkers in serum provides a more readily available option for investigating and understanding early equine pregnancy.

The Effect of a Seaweed-Derived Calcium Supplement on Gastric Juice pH in the Horse.

Low gastric pH for extended periods of time can increase the risk of gastric ulceration in horses. Therefore, nutritional interventions that buffer stomach acid may be helpful to decrease ulcer risk. The objective of this trial was to evaluate whether the incorporation of calcified Lithothamnion corallioides and Phymatolithon calcareum (Calmin; Celtic Sea Minerals, Cork, Ireland) into an equine ration would buffer equine gastric juice. Nine mature, Thoroughbred-cross horses, including 6 geldings and 3 mares (524 ± 49 kg) were housed in stalls and fed 2 kg/day of a texturized concentrate (Purina Omolene 100) and 1.5% BW grass hay/day. On testing days 0, 7, and 14, the horses received one of three pelleted dietary treatments (CON, MIN1 ×, MIN2 ×) in a randomized, crossover design. CON contained no added Calmin, MIN1 × provided Calmin at a 1 × concentration, and MIN2 × provided a 2 × dose. All horses underwent gastroscopy (Karl Storz, El Segundo, CA) prior to feeding the treatments, and at 2 and 4 hours postfeeding. Gastric juice was aspirated and pH measured using a benchtop pH meter (ThermoOrion pH Meter Model 410A). Overall, there was a significant time effect (P < .0001) with an increase in gastric juice pH from time 0 (2.31 ± 0.58) to 2 hours (5.52 ± 0.48) and 4 hours (3.59 ± 0.48). Gastric juice pH at 2 hours was higher (P = .0122) in MIN1 × (5.92 ± 0.58) and MIN2 × (5.92 ± 0.57) than CON (5.08 ± 0.58). These results demonstrate that adding Calmin to a meal increases buffering capacity at 2 hours postfeeding.

Influence of Metabolic Status and Diet on Early Pregnant Equine Histotroph Proteome: Preliminary Findings.

Insulin resistance (IR) is characterized by an increase in biomarkers of systemic inflammation and susceptibility to laminitis in horses. Impacts on reproduction include a lengthened interovulatory period in horses. Dietary omega-3 (docosahexaenoic acid [DHA]) promotes anti-inflammatory processes, has been implicated in health benefits, and can reduce cytokine secretion. This preliminary study investigated the impact of IR as well as the influence of dietary supplementation (DHA) on the uterine fluid proteome in early pregnant horses. Mares were artificially inseminated; uterine fluid and embryos were collected on d 12.5 after ovulation. Uterine fluid was pooled for metabolic and diet categories (n = 8; n = 2 per metabolic and dietary status) and concentrated, and the proteome was analyzed using tandem mass spectrometry (iTRAQ). Five proteins met differential abundance criteria (±1.5-fold change, P < .05) in all comparisons (Control C, IS vs. C, IR; C, IS vs. DHA, IS; C, IR vs. DHA, IR). Serum amyloid A, afamin, and serotransferrin were upregulated in C, IR mares but downregulated in DHA, IR mares when compared to C, IS and C, IR, respectively. Quantitative PCR supported mass spectrometry results. The presence of serum amyloid A and serotransferrin in histotroph of IR mares potentially indicates an inflammatory response not seen in IS counterparts. These preliminary findings provide novel evidence on the potential impact of insulin resistance and DHA supplementation on the secreted equine uterine proteome during early pregnancy.

Dietary tributyrin supplementation and submaximal exercise promote activation of equine satellite cells.

Postexercise skeletal muscle repair is dependent on the actions of satellite cells (SCs). The signal(s) responsible for activation of these normally quiescent cells in the horse remain unknown. The objective of the experiment was to determine whether submaximal exercise or tributyrin (TB) supplementation is sufficient to stimulate SC activation. Adult geldings were fed a control diet (n = 6) or a diet containing 0.45% TB (n = 6). After 30 d, the geldings performed a single bout of submaximal exercise. Middle gluteal muscle biopsies and blood were collected on days -1, 1, 3, and 5 relative to exercise. Diet had no effect on any parameter of physical performance. Total RNA isolated from the gluteal muscle of TB fed geldings contained greater (P < 0.05) amounts of myogenin mRNA than controls. Satellite cell isolates from TB supplemented horses had a greater (P = 0.02) percentage of proliferating cell nuclear antigen immunopositive (PCNA+) SC than controls after 48 h in culture. Submaximal exercise was sufficient to increase (P < 0.05) the percentage of PCNA(+) cells in all isolates obtained during recovery period. No change in the amount of gluteal muscle Pax7 mRNA, a lineage marker of SCs, occurred in response to either diet or exercise. Our results indicate that both submaximal exercise and TB prime SCs for activation and cell cycle reentry but are insufficient to cause an increase in Pax7 expression during the recovery period.

Tissue organization alters gene expression in equine induced trophectoderm cells.

Rapid morphological and gene expression changes occur during the early formation of a mammalian blastocyst. Critical to successful retention of the blastocyst and pregnancy is a functional trophectoderm (TE) that supplies the developing embryo with paracrine factors and hormones. The contribution of TE conformational changes to gene expression was examined in equine induced trophoblast (iTr) cells. Equine iTr cells were cultured as monolayers or in suspension to form spheres. The spheres are hollow and structurally reminiscent of native equine blastocysts. Total RNA was isolated from iTr monolayers and spheres and analyzed by RNA sequencing. An average of 32.2 and 31million aligned reads were analyzed for the spheres and monolayers, respectively. Forty-four genes were unique to monolayers and 45 genes were expressed only in spheres. Conformation did not affect expression of CDX2, POU5F1, TEAD4, ETS2, ELF3, GATA2 or TFAP2A, the core gene network of native TE. Bioinformatic analysis was used to identify classes of genes differentially expressed in response to changes in tissue shape. In both iTr spheres and monolayers, the majority of the differentially expressed genes were associated with binding activity in cellular, developmental and metabolic processes. Inherent to protein:protein interactions, several receptor-ligand families were identified in iTr cells with enrichment of genes coding for PI3-kinase and MAPK signaling intermediates. Our results provide evidence for ligand initiated kinase signaling pathways that underlie early trophectoderm structural changes.

Comparison of an in-helmet temperature monitor system to rectal temperature during exercise.

Body temperature monitoring is crucial in helping to decrease the amount and severity of heat illnesses; however, a practical method of monitoring temperature is lacking. In response to the lack of a practical method of monitoring the temperature of athletes, Hothead Technologies developed a device (HOT), which continuously monitors an athlete's fluctuations in body temperature. HOT measures forehead temperature inside helmets. The purpose of this study was to compare HOT against rectal temperature (Trec). Male volunteers (n = 29, age = 23.5 ± 4.5 years, weight = 83.8 ± 10.4 kg, height = 180.1 ± 5.8 cm, body fat = 12.3 ± 4.5%) exercised on a treadmill at an intensity of 60-75% heart rate reserve (HRR) (wet bulb globe temperature [WBGT] = 28.7° C) until Trec reached 38.7° C. The correlation between Trec and HOT was 0.801 (R = 0.64, standard error of the estimate (SEE) = 0.25, p = 0.00). One reason for this relatively high correlation is the microclimate that HOT is monitoring. HOT is not affected by the external climate greatly because of its location in the helmet. Therefore, factors such as evaporation do not alter HOT temperature to a great degree. HOT was compared with Trec in a controlled setting, and the exercise used in this study was moderate aerobic exercise, very unlike that used in football. In a controlled laboratory setting, the relationship between HOT and Trec showed favorable correlations. However, in applied settings, helmets are repeatedly removed and replaced forcing HOT to equilibrate to forehead temperature every time the helmet is replaced. Therefore, future studies are needed to mimic how HOT will be used in field situations.