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.

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.

Feeding Grass Hay Before Concentrate Mitigates the Effect of Grain-Based Concentrates on Postprandial Plasma Interleukin-1ß.

When fed to horses, high-starch diets elevate plasma concentrations of interleukin-1ß (IL-1ß) as soon as 1 hour posteating. This increase in IL-ß is possibly because of changes in intestinal pH that result from rapid bacterial fermentation of starches and sugars in the digestive tract. The purpose of this research was to investigate the effect of feeding 0.9 kg of grass hay 30 minutes before feeding a concentrate meal on the postprandial rise in IL-1ß, compared with control horses receiving the same concentrate without hay first. Six mature geldings were used in a switchback design. Horses were fasted overnight before being offered a concentrate feed that provided 1.2 g/kg bodyweight of nonstructural carbohydrates. Plasma was harvested 30 minutes before hay feeding and 1, 2, 4, 6, and 8 hours postfeeding. Concentrations of IL-1ß and d-lactate were analyzed by repeated measures analysis of variance. The hay-first treatment reduced (P = .034) postprandial concentrations of IL-1ß at all time points compared with the control horses. An interaction between hour and treatment was detected for mean d-lactate concentrations (P = .037), with lower concentrations in hay-first fed horses at postfeeding hours 1, 2, and 4, compared with control horses. Given these findings, we believe that feeding a small amount of hay before feeding a meal of moderate starch and sugar content reduced the negative effects of rapid starch and sugar fermentation in the equine digestive tract, evidenced by reduced postprandial d-lactate and IL-1ß concentrations.

Identification and validation of risk loci for osteochondrosis in standardbreds.

BACKGROUND: Osteochondrosis (OC), simply defined as a failure of endochondral ossification, is a complex disease with both genetic and environmental risk factors that is commonly diagnosed in young horses, as well as other domestic species. Although up to 50 % of the risk for developing OC is reportedly inherited, specific genes and alleles underlying risk are thus far completely unknown. Regions of the genome identified as associated with OC vary across studies in different populations of horses. In this study, we used a cohort of Standardbred horses from the U.S. (n = 182) specifically selected for a shared early environment (to reduce confounding factors) to identify regions of the genome associated with tarsal OC. Subsequently, putative risk variants within these regions were evaluated in both the discovery population and an independently sampled validation population of Norwegian Standardbreds (n = 139) with tarsal OC. RESULTS: After genome-wide association analysis of imputed data with information from >200,000 single nucleotide polymorphisms, two regions on equine chromosome 14 were associated with OC in the discovery cohort. Variant discovery in these and 30 additional regions of interest (including 11 from other published studies) was performed via whole-genome sequencing. 240 putative risk variants from 10 chromosomes were subsequently genotyped in both the discovery and validation cohorts. After correction for population structure, gait (trot or pace) and sex, the variants most highly associated with OC status in both populations were located within the chromosome 14 regions of association. CONCLUSIONS: The association of putative risk alleles from within the same regions with disease status in two independent populations of Standardbreds suggest that these are true risk loci in this breed, although population-specific risk factors may still exist. Evaluation of these loci in other populations will help determine if they are specific to the Standardbred breed, or to tarsal OC or are universal risk loci for OC. Further work is needed to identify the specific variants underlying OC risk within these loci. This is the first step towards the long-term goal of constructing a genetic risk model for OC that allows for genetic testing and quantification of risk in individuals.

Detection of prosecretory mitogen lacritin in nonprimate tears primarily as a C-terminal-like fragment.

PURPOSE: Lacritin is a human tear glycoprotein that promotes basal tear protein secretion in cultured rat lacrimal acinar cells and proliferation of subconfluent human corneal epithelial cells. When topically added to rabbit eyes, lacritin promotes basal tearing. Despite these activities on several species, lacritin's presence in nonprimate tears or other tissues has not been explored. Here we probed for lacritin in normal horse tears. METHODS: Sequences were collected from the Ensembl genomic alignment of human LACRT gene with high-quality draft horse genome (EquCab2.0) and analyzed. Normal horse tears were collected and assayed by Western blotting, ELISA, and mass spectrometry. Newly generated rabbit antibodies, respectively, against N- and C-terminal regions of human lacritin were employed. RESULTS: Identity was 75% and 45%, respectively, at nucleotide and protein levels. Structural features were conserved, including a C-terminal amphipathic α-helix. Anti-C-terminal antibodies strongly detected a ∼13 kDa band in horse tears that was validated by mass spectrometry. In human tears, the same antibody detected uncleaved lacritin (∼24 kDa) strongly and C-terminal fragments of ∼13 and ∼11 kDa weakly. Anti-N-terminal antibodies were slightly reactive with a ∼24 kDa horse antigen and showed no reaction with the anti-C-terminal-reactive ∼13 kDa species. Similar respective levels of horse C-terminal versus N-terminal immunoreactivity were apparent by ELISA. CONCLUSIONS: Lacritin is present in horse tears, largely as a C-terminal fragment homologous to the mitogenic and bactericidal region in human lacritin, suggesting potential benefit in corneal wound repair.

10.1093/jn/136.7.2090S

Treatment of clinical laminitis usually fails to prevent some degree of persistent disability; thus, intervention should aim at avoiding risk factors and preventing the disease. Efficiency of intervention would be improved by identifying predisposed horses and ponies. A herd of 160 healthy ponies included 54 previously laminitic (PL) and 106 never laminitic (NL). Pedigree analysis was consistent with dominant inheritance partially suppressed in males. Blood analysis revealed higher plasma concentrations of insulin and triglycerides but not cortisol, glucose, or free fatty acids in the PL group. Proxies for insulin sensitivity and beta-cell responsiveness, which were calculated from plasma insulin and glucose, indicated compensated insulin resistance in the PL group. A prelaminitic metabolic syndrome (PLMS) was derived statistically to have cut-off points for the 2 proxies, hypertriglyceridemia, and body condition score. It had a total predictive power of 78%. It identified 62 ponies with PLMS, and 98 as PLMS-free. Two months later, pasture starch concentration doubled, and 13 clinical cases of laminitis developed, 11 in the PLMS group and 2 in the PLMS-free group, giving an odds ratio of 10.4 (P = 0.0006). The PLMS can be used to identify predisposed ponies in need of special care; the efficiency of intervention would increase nearly 3-fold in the present case. It enables the design of new interventions suitable for testing. The PLMS also might influence market values.

Evaluation of genetic and metabolic predispositions and nutritional risk factors for pasture-associated laminitis in ponies.

OBJECTIVE: To evaluate genetic and metabolic predispositions and nutritional risk factors for development of pasture-associated laminitis in ponies. DESIGN: Observational cohort study. ANIMALS: 160 ponies. PROCEDURES: A previous diagnosis of laminitis was used to differentiate 54 ponies (PL group) from 106 nonlaminitic ponies (NL group). Pedigree analysis was used to determine a mode of inheritance for ponies with a previous diagnosis of laminitis. In early March, ponies were weighed and scored for body condition and basal venous blood samples were obtained. Plasma was analyzed for glucose, insulin, triglycerides, nonesterified fatty acids, and cortisol concentrations. Basal proxies for insulin sensitivity (reciprocal of the square root of insulin [RISQI]) and insulin secretory response (modified insulin-to-glucose ratio [MIRG]) were calculated. Observations were repeated in May, when some ponies had signs of clinical laminitis. RESULTS: A previous diagnosis of laminitis was consistent with the expected inheritance of a dominant major gene or genes with reduced penetrance. A prelaminitic metabolic profile was defined on the basis of body condition, plasma triglyceride concentration, RISQI, and MIRG. Meeting > or = 3 of these criteria differentiated PL- from NL-group ponies with a total predictive power of 78%. Determination of prelaminitic metabolic syndrome in March predicted 11 of 13 cases of clinical laminitis observed in May when pasture starch concentration was high. CONCLUSIONS AND CLINICAL RELEVANCE: Prelaminitic metabolic syndrome in apparently healthy ponies is comparable to metabolic syndromes in humans and is the first such set of risk factors to be supported by data in equids. Prelaminitic metabolic syndrome identifies ponies requiring special management, such as avoiding high starch intake that exacerbates insulin resistance.