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Nine pregnant mares (18.2 ± 0.7 yr; 493.82 ± 12.74 kg body weight [ BW ]) were used to test the hypothesis that dietary supplementation of l -arginine would enhance placental vascularity and nutrient transport throughout gestation in aged mares. Mares were balanced by age, BW, and stallion pairing, and assigned randomly to dietary treatments of either supplemental l -arginine (50 mg/kg BW; n  = 7) or l -alanine (100 mg/kg BW; n  = 6; isonitrogenous control). Mares were individually fed concentrate top-dressed with the respective amino acid treatment plus ad libitum access to Coastal Bermudagrass hay. Treatments began on day 14 of gestation and were terminated at parturition. Mare BW, body condition score (BCS), and rump fat were determined, and body fat percentage was calculated every 28 d and concentrate adjusted accordingly. Doppler blood flow measurements including resistance index (RI) and pulsatility index for uterine artery ipsilateral to the pregnant uterine horn were obtained beginning on day 21 and continued every 7 d until day 154 of gestation, and prior to parturition. Parturition was attended with foaling variables and placental measures recorded. Placental tissue from the pregnant horn was analyzed histologically to assess cell-specific localization of vascular endothelial growth factor (VEGF) and cationic amino acid transporter 1 (SLC7A1) proteins. Semiquantitative analyses were performed using 10 nonoverlapping images per sample fixed in a 10× field (Fiji ImageJ v1.2). Mare performance data were analyzed using PROC MIXED in SAS and foaling and placental data were analyzed using PROC GLM. Gestation length at parturition was not influenced (P  > 0.05) by supplemental arginine. Compared with arginine-supplemented mares, control mares had a thicker rump fat layer (P  < 0.01) and greater percent body fat (P  = 0.03), and BCS (P  < 0.01) at parturition. Arginine-supplemented mares had a lower RI than control mares prior to parturition (P  < 0.01). Body length, height, and BW of foals at birth, as well as placental weight and volume, and immunohistochemical staining for VEGF and SLC7A1 at parturition, were not affected (P  > 0.05) by maternal arginine supplementation. These results indicate that dietary arginine supplementation (50 mg/kg BW) is safe for gestating mares. A larger number of mares is required to extend knowledge of effects of supplemental arginine on embryonic/fetal survival and growth in mares. [ABSTRACT FROM AUTHOR]

Bisphosphonates alter bone metabolism via inhibition of osteoclastic bone resorption. Although bisphosphonates are beneficial to treat bone disease, extra-label use in healthy juvenile horses may interfere with normal bone metabolism. The objective was to determine plasma pharmacokinetics of the bisphosphonate clodronate disodium (CD) following a single intramuscular dose to better define the time course of CD in juvenile horses. Ten yearling Quarter Horses (334 ± 18 kg, 504 ± 13 d of age) were used to test the hypothesis that juvenile horses would have faster clearance rate of CD compared with previously published data in adult horses. Horses were placed in individual stalls, jugular intravenous catheters were inserted, and an initial blood sample (0 h) was collected. Horses were administered CD intramuscularly at 1.8 mg/kg BW per label. Plasma samples were collected at 0.5, 1, 3, 6, 12, 24, 48, and 72 h post-administration. Plasma concentration of clodronate was determined by liquid chromatography tandem mass spectrometry. Pharmacokinetic parameters were calculated using a two-compartment model. Blood chemistry parameters [blood urea nitrogen (BUN), creatinine, alkaline phosphatase (ALKP), aspartate aminotransferase (AST), gamma glutamyl transferase (GGT), and creatine kinase (CK)] were determined via a commercial laboratory. Included blood chemistry parameters were analyzed for an effect of time using PROC Mixed of SAS. Clodronate was detected in plasma up to 72 h post-administration. Maximum plasma concentration (43,971 ± 25,920 ng/mL) was reached at 0.5 h postadministration. Analysis revealed bicompartmental kinetics with distribution and terminal half-lives of 1.9 ± 0.4 h and 24.4 ± 4.3 h, respectively. Area under the curve to last sample was 22,755 ± 11,426 ng*h/mL and extrapolated to infinity was 26,778 ± 12,465 ng*h/mL. Blood chemistry parameters varied over time within normal ranges. Markers of renal function, BUN and creatinine, decreased from h 0 to h 72 (P < 0.01). Liver function markers ALKP and AST were not affected by CD treatment (P > 0.27). However, GGT and CK changed over time (P < 0.01), with GGT greater at 48 h than other time points, and CK increasing from 0.5 to 24 h before decreasing by 72 h but remaining above baseline. Plasma clodronate concentrations were greater in juvenile horses than previously reported in mature horses (Krueger et al., 2020, Equine Vet J.; Knych et al., 2022, Equine Vet J.). Further, the observed bicompartmental kinetics differed from the linear kinetics previously reported in mature horses whereby juvenile horses from the current study had greater exposure to CD over time as indicated by a longer terminal half-life. Moreover, the single intramuscular dose of CD (1.8 mg/kg BW) did not cause markers of kidney or liver function to deviate from normal ranges in juvenile Quarter Horses. [ABSTRACT FROM AUTHOR]

Including Saccharomyces cerevisiae fermentation product (SCFP) into the diets of young, exercising horses may prolong their performance career by optimizing the intra-articular environment. Our objective was to evaluate the effect of dietary SCFP on joint inflammation in yearlings undergoing regular exercise, challenged with intra-articular lipopolysac-charide (LPS). Thirty Quarter Horse yearlings (374 ± 25 kg BW; 562 ± 16 d of age; 15 fillies and 15 geldings) were used in a 60-d study to test the hypothesis that dietary SCFP (TruEquine C, Diamond V Mills, Inc.) ameliorates joint inflammation following an acute inflammatory insult. Horses were stratified by BW, age, sex, and randomly assigned to dietary treatments (n = 10/treatment): Control (0), 46, or 92 mg/kg BW SCFP. Treatments were top-dressed onto a custom-formulated concentrate void of added microbials offered at 1% BW/d (as-fed) every 12 h. Horses were individually stalled (3.6 m x 7.3 m) and offered ad libitum Coastal bermudagrass hay. Using a free-stall exerciser, horses were exercised following a progressive workload regimen for 30 min/d, 5 d/wk. On d 46, all horses underwent an intra-articular LPS challenge where each horse had one radial carpal joint randomly assigned to receive either 0.8 mL of a 0.5 ng LPS solution or sterile lactated Ringer's solution (LRS) as a contralat-eral control. Synovial fluid samples were obtained pre-injection (h 0) and at 6, 12, 24, and 336 h post-injection and analyzed for prostaglandin E2 (PGE2), a proinflammatory prostaglandin via commercial ELISA, and for chemokine-concentration (signaling proteins that direct immune cells; CCL2, and CCL11) and cytokines (inflammatory mediators; TNFα and IL-10) using a multiplex platform via commercial laboratory. Non-normal data (PGE2, IL-10, and CCL2) were log transformed, and all markers were analyzed using MIXED procedure of SAS v9.4. Mean separation was achieved with orthogonal contrasts, and contralateral control carpi were used as a covariate across all hours. There was no effect of SCFP on synovial logPGE2 (P = 0.42), logCCL2 (P = 0.90), CCL11 (P = 0.26), or logIL-10 (P = 0.23). However, there was a treatment x hour interaction for CCL11 (P = 0.04) where the Control had increased concentrations compared with SCFP treatment groups at 6 h post-injection. Furthermore, logIL-10 also had a treatment x hour interaction (P = 0.05) where the 46mg/kg BW group had decreased concentrations at h 12 compared with the Control and 92mg/kg BW group. The main effect of treatment for TNFa (P = 0.04) revealed that the 92mg/kg BW group had less concentrations than the 46mg/kg BW group and tended to have less concentrations than the Control group across all hours. Results indicate that SCFP may help mitigate inflammation markers following an acute intra-articular inflammatory insult. [ABSTRACT FROM AUTHOR]

Stock-type mares (498 ± 9 kg BW; 12 ± 7 yr) were used in a completely randomized design for 56 d to test the hypothesis that concentrate fortification improves apparent digestion and enhances lean mass over the topline. Horses were stratified by age, BW, and BCS and randomly assigned to either a custom pelleted concentrate (CON; n = 13), or an iso-caloric, iso-nitrogenous pellet that included amino acid fortification, complexed trace minerals, and fermentation metabolites (FORT; n = 10). Concentrate was offered at a total 0.75% BW/d (as-fed) twice daily, and diets were designed to meet or exceed maintenance requirements for mature horses. Horses had ad libitum access to Coastal bermudagrass hay (7.4% CP, 67% NDF, and 40% ADF). Every 14 d BW and BCS were recorded, and ultrasound images were captured every 28 d. longissimus dorsi area (LDA) and subcutaneous fat thickness (FT) were measured between the 12th and 13th ribs (12th/13th) and 17th and 18th ribs (17th/18th). Intramuscular fat at the 17th/18th ribs and rump fat-thickness were also obtained. Horses were dosed with 10 g/d of titanium dioxide (TiO2) for 14 d to estimate forage dry matter intake (DMI). To account for diurnal variation, fecal samples were collected twice daily at 12-h intervals during the last 4 days, advancing by 3 h each day to represent a 24-h period. Fecal samples were composited by horse and analyzed for TiO2 to estimate fecal output and acid detergent insoluble ash was used to calculate forage DMI. To evaluate body composition, horses were infused with a 0.12 g/kg BW deuterium oxide (D2O) on d 0 and 56. Body fat percentage (BF) was determined by quantifying D2O in plasma samples collected at pre- and 4-h postinfusion via mass spectrometry. All data were analyzed using PROC MIXED (SAS v9.4). The model contained a fixed effect of diet; horse (diet) was a random effect. Horses receiving FORT gained 17th/18th FT (P < 0.01) and increased 17th/18th LDA from d 0 to 56 (P < 0.01) while 17th/18th FT and LDA were unchanged in CON. Regardless of diet, BF estimated by D2O infusion increased in all horses from d 0 to 56 (P < 0.01). Average hay DMI was 2.1% BW, but did not differ between diets. In this study, concentrate fortification did not significantly (P ≥ 0.27) affect apparent digestion. In conclusion, concentrate fortification may promote greater muscle development along the posterior topline. [ABSTRACT FROM AUTHOR]

Passive immune transfer in a newborn foal is highly dependent upon the quality and concentration of immunoglobulins (Ig) in maternal colostrum, primarily IgG. Anti-endotoxin IgY derived from chicken egg yolks has been used in calves to reduce morbidity and mortality. Its use may provide additional pathogen-specific immune protection to neonatal foals, but passive transfer has not been confirmed. Therefore, the objective of this study was to quantify passive IgY transfer in neonatal foals given an egg-derived anti-endotoxin IgY supplement (Camas, Inc.) and identify effects on passive immunity. To test this, 5 neonatal foals were randomly divided into two treatment groups. Within 20 minutes after first nursing, the treated group (TRT; n = 3) was administered 40 g of the egg yolk supplement diluted in 50 mL water orally via syringe and the control group (CON; n = 2) was given isovolumetric water. At birth, physical measurements including BW, wither height (WH), hip height (HH), body length (BL), and heart girth (HG) circumference were recorded. Serum samples were collected prior to first nursing (h 0) and h 2, 6, 12, 24, 72, 168, 336 post treatment administration. Rectal temperature, heart rate, and respiration rate were recorded at all blood collection timepoints. Serum samples were analyzed for IgG, IgA, and IgY by commercial ELISA. Custom ELISA was used to identify endotoxinspecific IgY for Salmonella and E. coli at h 6. Western immunoblotting was used to confirm that passively transferred IgY was intact. Data were analyzed using PROC MIXED in SAS. Vital signs remained within normal, healthy ranges and physical measurements at birth were similar for all foals (P > 0.26). There was an effect of treatment (P = 0.05), time (P < 0.01), and treatment × time interaction (P < 0.01) for serum IgY concentrations where IgY was only detectable in TRT foals and peaked at h 6 and 12, decreasing thereafter to h 336. Concentrations of IgG and IgA increased from h 0 to 12 (P < 0.01); however, there were no effects of treatment nor treatment × time interactions (P > 0.24) for either Ig. Western immunoblotting confirmed that IgY was transferred intact and custom ELISA identified IgY specific against Salmonella and Escherichia coli. These results confirm the intact passive transfer of IgY in neonatal foals when given an IgYinfused egg yolk supplement with no negative impact on passive transfer of IgG and IgA from maternal colostrum. [ABSTRACT FROM AUTHOR]

Anecdotal assertions that bisphosphonates, such as clodronate disodium (CD), exhibit anti-inflammatory properties has led to extra-label use, despite a lack of scientific evidence of its efficacy. The objective of this study was to determine the effects of CD on intra-articular inflammation, hypothesizing that intra-muscular administration of CD would reduce intra-articular inflammation following an acute inflammatory challenge by lipopolysaccharide (LPS). To test this, 32 yearling Quarter horses were stratified by age (500 ± 13 d), BW (336 ± 26 kg), sex (n = 16 female; n = 16 male) and initial bone optical density into 4 treatment groups. The 140-d study consisted of two phases: Phase 1 (d 0 to 83) and Phase 2 (d 84 to 140) emulated sale preparation and early performance training, respectively. Horses were housed individually (3.6 m × 7.3 m) and fed to meet requirements of growing horses undergoing moderate training. Treatments consisted of control (CON; n = 8; no CD), single-dose (1X; n = 8; d 84), two-doses (2X; n = 8; d 0, 84), and four-doses (4X; n = 8; d 0, 42, 84, 126) of CD. All horses received isovolumetric intramuscular injections of 1.8mg/kg BW clodronate disodium (OSPHOS) or saline (placebo) on d 0, 42, 84, and 126. Following treatment administration on d 126, radial carpal joints of each horse were injected with 0.8 mL of either 0.5 ng sterile LPS derived from Escherichia coli O55:B5 or sterile lactated Ringer's solution as a contralateral control. Synovial fluid was collected before LPS injection (h 0) and at 6, 12, 24, and 336 h post injection. Synovial fluid was analyzed using an ELISA for prostaglandin E2 (PGE2), an indicator of joint inflammation. Data were log transformed and analyzed using the MIXED procedure of SAS. There was an h × LPS interaction (P < 0.01) confirming an acute, transient inflammatory response with increased PGE2 at 6, 12, and 24 h compared with the contralateral control joint, and resolved (P = 0.62) by h 336 post-injection. There was a treatment × h × LPS interaction (P = 0.02) in which 1X, 2X and 4X CD groups had greater synovial PGE2 concentrations at 6 h post-injection in the LPS joint compared with CON. In summary, intra-articular LPS induced localized inflammation, and the PGE2 response was greater in all horses treated with CD. The results of this study reject the hypothesis, suggesting clodronate disodium administration does not reduce intra-articular inflammation following acute induced synovitis. [ABSTRACT FROM AUTHOR]

Undernutrition is common for animals maintained in a range setting, particularly during periods of drought, and during gestation can be detrimental to offspring development. The placenta is a known mediator for maternal-fetal nutrient exchange, and developmental impacts to the placenta during times of nutritional stress contribute to poor offspring outcomes. The objective of this study was to determine the impacts of maternal nutrient restriction on the development and function of placental cotyledons. Targhee ewes were allocated into two dietary treatment groups for the entirety of gestation. The control group (CON; n = 10) was fed to meet 100% of NRC requirements and the nutrient restricted group (NR; n = 11) was fed to meet 60% of NRC requirements, to mimic the nutritional stress experienced by range ewes during drought. At the time of lambing total placental weight, total cotyledon number, and lamb birth weight were recorded. Cotyledons (n = 3 / placenta) were removed from multiple locations, weighed, and processed for RNA-sequencing. Total placental weight, total cotyledon number, and lamb birth weight were not affected by maternal diet restriction (P > 0.05). Average cotyledon weight was greater in the CON group compared with the NR group (P < 0.05). Gene ontology (GO) and Kyoto encyclopedia of genes and genomes pathways (KEGG) analyses were performed to identify biological pathways involved in placental function and nutrient transfer. Cotyledons from NR singletons vs CON singletons expressed 470 differentially expressed genes (DEGs; P < 0.05; 187 up-regulated, 283 down-regulated). Gene ontology terms represented by DEGs include hormone activity (P = 0.0003) and antigen processing and presentation (P = 0.0004). KEGG pathways of interest include pyruvate metabolism, estrogen signaling, extracellular matrix receptor interaction, and antigen processing and presentation. There were also 426 DEGs (P < 0.05; 195 up-regulated, 231 down-regulated) in cotyledons of NR twins vs CON twins. Represented GO categories include heme binding (P = 0.003) and oxidoreductase activity (P = 0.03). These data show that prolonged nutrient restriction alters cotyledon development and gene expression indicating modifications in placental function. These changes in placental function likely mediate poor offspring developmental outcomes observed following gestational under nutrition. [ABSTRACT FROM AUTHOR]

Off-label bisphosphonate use in juvenile horses is widespread despite little scientific understanding of biological and welfare impacts on skeletally immature, exercising horses. Therefore, the objective was to determine the effects of clodronate disodium on growth parameters of young horses. Thirty-two yearling Quarter horses (335 ± 4 kg, 500 ± 13 d of age) were stratified by age, BW, sex, and initial bone density by aluminum equivalence into four treatment groups for a 168-d trial. The experimental period was divided into two phases mimicking common management practices in horses undergoing sales preparation (Phase I: d 0 to 83) and early performance training (Phase II: d 84 to 168). Horses were housed in individual stalls (3.6 m × 7.2 m). Diets were formulated to meet nutrient requirements including concentrate offered every 12 h and ad libitum access to coastal bermudagrass (Cynodon dactylon) hay and water. Treatments consisted of control (CON; n = 8), single-dose (1X; n = 8), two-dose (2X; n = 8), and four-dose groups (4X; n = 8). All horses received iso-volumetric intramuscular injections of either 1.8 mg/kg BW clodronate disodium (OSPHOSÒ) or saline (placebo) on d 0, 42, 84, and 126. Physical measurements including BW, wither height (WH), hip height (HH), body length (BL), and heart girth (HG) circumference were recorded every 42 d, beginning on d 0. Data were analyzed using PROC MIXED procedure of SAS. There were no treatment differences (P = 0.62) or treatment' time interactions (P = 0.25) for BW, WH, HH, BL, and HG, but all measurements increased over time (P = 0.01). These results indicate that clodronate does not impact physical growth parameters in juvenile horses undergoing forced exercise. Further work is ongoing to determine tissue-specific effects of clodronate on bone growth and development in yearling Quarter horses. [ABSTRACT FROM AUTHOR]

Nine pregnant mares (18.2 ± 0.7 y; 493.82 ± 13 kg BW) were used to test the hypothesis that dietary supplementation of arginine would enhance placental vascularity and nutrient transport throughout gestation in aged mares. Mares were balanced by age, BW, and stallion pairing, and randomly assigned to one of two dietary treatments consisting of supplemental L-arginine (50 mg/kg BW; n=5) or L-alanine (100 mg/kg BW; n=4; isonitrogenous control). Mares were individually fed concentrate top-dressed with the respective amino acid treatment plus ad libitum access to Coastal bermudagrass hay. Treatments began on d 14 of gestation and were terminated at parturition. Mare BW, BCS, and rump fat were determined every 28 d and concentrate adjusted accordingly. Parturition was attended with foaling variables and placental measures recorded. Placental tissue from the pregnant horn underwent histological analyses to assess cell-specific localization of vascular endothelial growth factor (VEGF) and cationic amino acid transporter 1 (CAT1) proteins. Semiquantitative analyses were performed using 10 non-overlapping images per sample fixed in a 10X field (Fiji ImageJ v1.2). Data were analyzed using SAS PROC MIXED procedure. BW increased (P < 0.01) in all mares with advancing gestation. BW, BCS, and gestation length of mares were not influenced (P > 0.05) by supplemental arginine. Compared with argininesupplemented mares, control mares had a thicker rump fat layer (P < 0.01) at parturition. Body length, height, and BW of foals at birth, as well as placental weight, volume, and positive staining for VEGF and CAT1 at parturition were not affected (P > 0.05) by maternal arginine supplementation. These results indicate dietary arginine supplementation (50 mg/kg BW) is safe for gestating mares and promotes mobilization of white adipose tissue. A larger number of mares is required to determine effects of supplemental arginine on embryonic/fetal survival and growth in mares. [ABSTRACT FROM AUTHOR]