L.M. Lawrence

Effect of body condition, body weight and adiposity on inflammatory cytokine responses in old horses

Advanced age is associated with a low-grade, systemic inflammatory response characterized by increased inflammatory cytokine production both in vitro and in vivo, termed inflamm-aging. It is also known that increased white adipose tissue, associated with obesity, leads to increased production of inflammatory cytokines. To date, it is unknown whether increased adiposity contributes to the age-related increased inflammatory status. Here we show that peripheral blood mononuclear cells (PBMC) from old horses compared to young horses have increased inflammatory cytokine production; moreover, fat old horses compared to thin old horses have even greater frequencies of lymphocytes and monocytes producing inflammatory cytokines. Therefore, we proposed that decreasing adiposity in old horses would reduce age-associated increases of inflammatory cytokines both in vitro and in vivo, and increasing adiposity in old horses would increase these measurements. To test this hypothesis further, eight old obese horses (20-28 year) were assigned to two consecutive treatments, dietary restriction (DR) during weeks 1-12 and increased dietary intake (DI) during weeks 13-30. Body weight, body condition score (BCS) and percent body fat were measured weekly. PBMC were stimulated in vitro and interferon gamma (IFNgamma) and tumor necrosis factor alpha (TNFalpha) production was measured by intracellular staining. Levels of nascent IFNgamma and TNFalpha mRNA expression were examined by RT-PCR. Serum concentrations of TNFalpha protein were also measured weekly. Reducing body weight and fat in old horses significantly reduced the percent of IFNgamma and TNFalpha positive lymphocytes and monocytes, and serum levels of TNFalpha protein. Further, when weight and fat increased in these old horses there was a significant increase in inflammatory cytokine production. Regression analysis also revealed significant relationships. These findings demonstrate that age-related obesity potentially plays a role in the dysregulation of inflammatory cytokine production by the immune system with age or inflamm-aging in the horse.

Diarrhea-associated pathogens, lactobacilli and cellulolytic bacteria in equine feces: Responses to antibiotic challenge

Antibiotics are important to equine medicine, but antibiotic-associated diarrhea (AAD) can lead to poor performance and even mortality. AAD is attributed to disruption of the hindgut microbiota, which permits proliferation of pathogenic microbes. The goal of this study was to evaluate the effects of common antibiotics on cellulolytic bacteria, lactobacilli, and AAD-associated pathogens in the feces of healthy horses. Fifteen horses were assigned to three treatment groups (blocked by age and sex): control (no antibiotics), trimethoprim-sulfadiazine (PO), or ceftiofur (IM). Fecal samples (n=8 per horse) were taken during dietary adaptation (3 weeks), antibiotic challenge (1 week), and withdrawal (1 week). Bacteria were enumerated by serial dilution and viable count. Cellulolytic bacteria decreased by >99% during administration of either antibiotic (P<0.0001) and were still less than controls at the end of the withdrawal period (P<0.0001). Fecal samples from horses challenged with ceftiofur had 75% fewer lactobacilli than those from control horses at the end of the antibiotic challenge period (P<0.05). Antibiotic challenged horses also shed more salmonella than control horses (P<0.05). Antibiotics had no effect on the number of Clostridium perfringens isolates. There was no detectable Clostridium difficile during adaptation or in any control horse. C. difficile increased (P<0.0001) to approximately 10(4)cfu/g when horses were challenged with antibiotics, and were still detectable 1 week after withdrawal. These results indicate that antibiotics can disrupt the normal gastrointestinal microbiota and allow proliferation of Salmonella spp. and C. difficile.

Comparison of in vitro digestibility estimates using the DaisyII incubator with in vivo digestibility estimates in horses.

The objective of this study was to determine if in vitro methodologies developed for the Ankom Daisy(II) incubator could produce accurate estimates of in vivo equine DM digestibility (DMD) and NDF digestibility (NDFD) when equine feces were used as the inoculum source. Four mature geldings were utilized in a 4 × 4 Latin square design experiment with a 2 × 2 factorial arrangement of dietary treatments (timothy hay, alfalfa hay, timothy hay plus oats, and alfalfa hay plus oats), in which the geldings were individually housed and fed. During each 5-d total fecal collection period, feces were collected and composited daily and used to calculate in vivo digestibility. Digestion of the 4 treatment diets was evaluated in vitro using the Daisy(II) incubator. Each incubation vessel of the Daisy(II) was assigned to 1 of the horses and contained 18 filter bags (6 containing the assigned treatment hay, 6 containing hay-oat mix, and 6 containing oats). Three incubation periods were evaluated: 30, 48, and 72 h. Although the 30- and 48-h in vitro estimates were consistently less than the in vivo estimates, they ranked diets in the same order as the in vivo method. For the alfalfa oat diet, timothy diet, and the timothy oat diet, the mean 72-h in vitro DMD and in vivo DMD were not different (P = 0.1444). However, for the alfalfa diet, the DMD estimate from 72-h in vitro incubation was less than the in vivo estimate (P < 0.010). For NDFD, the timothy diet was the only diet, in which the mean 72-h in vitro NDFD estimate was not different than the in vivo estimate. However, the in vitro method correctly ranked the alfalfa-based diets as having greater NDFD estimates than the timothy-based diets. Of the 3 incubation periods, the 72-h period provided digestibility estimates most similar to the in vivo data. Using the methodologies described in this research, the Daisy(II) incubator and equine feces can be used to estimate in vivo DMD of horse feeds.

Variation in the distribution of muscle fiber types in equine skeletal muscles

Summary Four skeletal muscles, semitendinosus (Se), longissimus dorsi (Lg), soleus (Su) and gluteus medius (Gm) were removed from a four-year-old female Welsh pony. The muscles were divided into sections which were histochemically examined to determine the variation in the distribution of fast-twitch glycolytic (FG), fast-twitch oxidative glycolytic (FOG) and slow-twitch oxidative (SO) muscle fibers throughout the entire cross section of the muscle. Within both the Se and Gm muscles significant variations were found in the distribution of all three fiber types. The percent of each fiber type differed amongall four adult skeletal muscles. This study demonstrated that there is significant variations in the distribution of the muscle fiber types within an individual muscle.

Inhibition of fructan‐fermenting equine faecal bacteria and Streptococcus bovis by hops (Humulus lupulus L.) β‐acid

The goals of this study were to determine if β‐acid from hops (Humulus lupulus L.) could be used to control fructan fermentation by equine hindgut micro‐organisms, and to verify the antimicrobial mode of action on Streptococcus bovis, which has been implicated in fructan fermentation, hindgut acidosis and pasture‐associated laminitis (PAL) in the horse.

Vasoconstriction in horses caused by endophyte-infected tall fescue seed is detected with Doppler ultrasonography.

The hypotheses that endophyte (Neotyphodium coenophialum)-infected tall fescue (TF) seed causes vasoconstriction in horses in vivo and that ground seed would cause more pronounced vasoconstriction than whole seed were tested. Ten horses each received 1 of 3 treatments: endophyte-free ground (E-G; n = 4 horses) seed, endophyte-positive whole (E+W; n = 3) seed, or endophyte-positive ground (E+G; n = 3) seed. There were two 14-d periods, P1 and P2. During P1, animals were adapted to a concentrate (0.2% BW, as fed, twice daily) and alfalfa cubes. During P2, the seed was mixed into the concentrate portion of the diet and alfalfa cubes were offered ad libitum. Fescue seed was fed in increasing amounts ranging from 0.02% BW on d 1 (averaging 76 ug/kg ergovaline + ergovalinine) to 0.22% BW on d 11 to 14 (averaging 713 ug/kg ergovaline + ergovalinine). The distal palmar artery of the left foreleg of each horse was scanned via Doppler ultrasonography for 4 d during each period, with 5 replicate scans performed on each scanning day. The measurements taken at each scan included artery luminal diameter, area, and circumference, peak systolic velocity, end diastolic velocity and blood flow variables. Animal temperature, heart rate, and respiration rate and ambient temperature and humidity were also recorded. Blood samples were taken on each scanning day to measure inflammatory cytokine mRNA abundances, and blood samples were collected on d 0, 4, 8, and 14 of P2 to measure prolactin concentrations. Consumption of E+G TF seed caused decreased artery lumen diameter (P = 0.0033), area (P = 0.0406), and circumference (P = 0.0480) compared with E-G seed, and E+W seed produced an intermediate response. Blood flow volume was reduced (P < 0.05) during P2 in horses receiving E+G seed compared with horses receiving E-G seed. Other ultrasound variables were not different (P > 0.05) among treatment groups, and neither were cytokine mRNA or prolactin concentrations. Treatment did not alter (P > 0.05) animal temperature, heart rate, or respiration rate, and neither ambient temperature nor relative humidity was consistently correlated with any response variable measured. Taken together, these data confirm that consumption of E+G fescue seed caused vasoconstriction in horses, which could be readily measured by Doppler ultrasonography. Use of Doppler ultrasound to monitor the diameter of the palmar artery of horses grazing endophyte-infected (E+) fescue pastures may provide a convenient and satisfactory biomarker to determine premonitory signs of fescue toxicosis.

Effect of Dietary Starch Source and Concentration on Equine Fecal Microbiota

Starch from corn is less susceptible to equine small intestinal digestion than starch from oats, and starch that reaches the hindgut can be utilized by the microbiota. The objective of the current study was to examine the effects of starch source on equine fecal microbiota. Thirty horses were assigned to treatments: control (hay only), HC (high corn), HO (high oats), LC (low corn), LO (low oats), and LW (low pelleted wheat middlings). Horses received an all-forage diet (2 wk; d -14 to d -1) before the treatment diets (2 wk; d 1 to 14). Starch was introduced gradually so that horses received 50% of the assigned starch amount (high = 2 g starch/kg BW; low = 1 g starch/kg BW) by d 4 and 100% by d 11. Fecal samples were obtained at the end of the forage-only period (S0; d -2), and on d 6 (S1) and d 13 (S2) of the treatment period. Cellulolytics, lactobacilli, Group D Gram-positive cocci (GPC), lactate-utilizers and amylolytics were enumerated. Enumeration data were log transformed and analyzed by repeated measures ANOVA. There were sample day × treatment interactions (P < 0.0001) for all bacteria enumerated. Enumerations from control horses did not change during the sampling period (P > 0.05). All treatments except LO resulted in increased amylolytics and decreased cellulolytics, but the changes were larger in horses fed corn and wheat middlings (P < 0.05). Feeding oats resulted in increased lactobacilli and decreased GPC (P < 0.05), while corn had the opposite effects. LW had increased lactobacilli and GPC (P < 0.05). The predominant amylolytic isolates from HC, LC and LW on S2 were identified by 16S RNA gene sequencing as Enterococcus faecalis, but other species were found in oat fed horses. These results demonstrate that starch source can have a differential effect on the equine fecal microbiota.

THERMAL ENVIRONMENT IN A FOUR-HORSE SLANT LOAD TRAILER

Little information has been published describing thermal conditions in horse trailers while in transit. Dry bulb temperature (Tdb), globe temperature (Tg), and relative humidity (RH) were measured in ten locations within a fully enclosed four-horse slant-load trailer with and without animals to assess the thermal environment during transport as influenced by vehicle speed and vent configuration. Wet bulb globe temperature (WBGT) was calculated to assess thermal comfort. Interior-exterior temperature differences were analyzed to account for effects of changing weather conditions. Temperature differences between the interior of the trailer and ambient conditions for Tdb ranged from 5.1°C to 9.5°C, dew point (Tdp) ranged from 4.4°C to 13°C, and WBGT ranged from 2.9°C to 7.9°C. Temperature differences decreased with increasing vehicle speed and open vent area and increased with animals present. Heat stress conditions are likely to occur in horse trailers of similar design given their limited ventilation and the temperature increases measured in this study, and warrant improvements in trailer design to increase ventilation.

A comparison of growth rates of normal thoroughbred foals and foals diagnosed with cervical vertebral malformation

SUMMARY The objectives of this study were to compare the growth rates of normal thoroughbred foals to previously reported growth rates 1 and to compare growth rates of normal foals with foals diagnosed with cervical vertebral malformation (CVM). Fifty-six foals from two foals crops were used, and eight of these foals were diagnosed with CVM. Growth rate analysis of the CVM foals had to be restricted to the period prior to diagnosis because once these foals were diagnosed, they were treated differently from the normal foals. Body measurements taken at 7 day intervals were body weight, wither height, hip height, and heart girth. For statistical analysis, data were broken down into 30 day intervals. Results showed that the growth rates for normal thoroughbred foals have not changed in the past fifteen years. The CVM foals tended to be heavier and taller during some time intervals than the normal foals, but there were no significant differences between the two groups in any of the skeletal growth measurements. Body weight gain was faster in CVM foals from 31–60 d (p

Measures of antioxidant status of the horse in response to selenium depletion and repletion.

Selenium plays a role in the antioxidant mechanism via the selenoenzyme glutathione peroxidase (GSH-Px). Change in Se status because of Se depletion or supplementation is associated with a change in GSH-Px activity and could potentially affect antioxidant status. This study evaluated the impact of change in Se status on measures of antioxidant status and oxidative stress in adult horses. Twenty-eight horses were blocked by age and gender and were randomly allocated to 1 of 4 dietary treatment groups: low Se (LS), adequate Se (AS), high organic Se (SP), and high inorganic Se (SS). For 196 d, LS, SP, and SS received a low-Se diet (0.06 mg Se/kg DM) to allow for depletion of Se stores, whereas AS received an adequate Se diet (0.12 mg Se/kg DM). Then, for the next 189 d, LS and AS were maintained on the same diets, whereas SP was supplemented with Se-yeast and SS with sodium selenite to allow for a total dietary Se intake of 0.3 mg Se/kg DM. Blood samples were collected throughout the study. Variables of interest included whole blood Se and GSH-Px activity, serum vitamin E concentration, total antioxidant capacity (TAC), serum malondialdehyde (MDA), and triiodothyronine and thyroxine concentrations. Data were analyzed using ANOVA with repeated measures. Whole blood Se and GSH-Px activity decreased in LS, SP, and SS during the depletion phase and increased in SP and SS with supplementation (treatment × time, P < 0.001). At the conclusion of the supplementation period, GSH-Px activity was greater in SP and SS compared with AS and LS (P < 0.05). Vitamin E status remained adequate throughout the study, and no differences existed between treatments. Serum TAC did not change in response to Se depletion or repletion. Serum MDA was greater for AS than LS during depletion (P < 0.05) but similar across treatments after supplementation. Overall, change in Se status did not have a large impact on TAC or MDA, possibly because the horses maintained an adequate vitamin E status. However, Se supplementation at 0.3 mg/kg DM increased GSH-Px activity above that of the horses fed an adequate diet based on the 2007 NRC recommendations, indicating a potential benefit to feeding greater Se diets to horses kept in low-Se areas.