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ANIMAL NUTRITION |
Department of Animal Sciences, University of Missouri, Columbia 65211
| Abstract |
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Key Words: Fecal Fructooligosaccharide Horse Microbiology Volatile Fatty Acids
| Introduction |
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Measurement of cecal or colonic responses, such as pH, microflora, short-chain fatty acid concentration, and epithelial cell proliferation, have been used to determine gut health in a number of species (Howard et al., 1995
; Campbell et al., 1997
; Garret et al., 2002). Fecal responses of pH, microflora, and color also have been used to assess colonic health in rats, pigs, and humans (Benno and Mitsuoka, 1992
; Howard et al., 1995
; Campbell et al., 1997
). Maintenance of colonic health in the horse is essential to avoid disorders such as colic, equine dysautonomia, or laminitis. Equine intestinal disorders in the United States (from spring 1998 to spring 1999) had an estimated cost of $115,300,000 (Traub-Dargatz et al., 2001
). Improving gut health in the horse has the potential to decrease this cost. Therefore, the objective of the present study was to compare the effects of two levels of FOS on the fecal responses of pH, microbial content, and VFA concentrations as indicators of intestinal health of yearling horses.
| Materials and Methods |
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Nine quarter horses (six geldings and three fillies) ranging in age from 489 to 539 d with initial BW averaging 400.6 ± 21.2 kg were used for this experiment. Horses were kept on an 8.1-ha orchardgrass pasture at the University of Missouri Horse Teaching and Research Facility and had ad libitum access to pasture grass, fresh water, and a plain salt block throughout the study. Horses were individually fed a concentrate supplement (Table 1
) at 1% of their BW on an as-fed basis daily according to NRC requirements (1989)
for long yearlings not in training, with the remaining requirements being met from pasture. This was a 30-d study conducted during September and October. The amount of concentrate fed was split equally between a morning (0730) and afternoon (1630) feeding, with the FOS being added daily to the morning feeding. The yearlings were vaccinated against Eastern and Western equine encephalitis, West Nile, tetanus, influenza, and rhinopneumonitis Types 1 and 4. The yearlings were dewormed every 2 mo with alternating anthelmintic products (January and July with fenbendazole, March and September with ivermectin, and May and November with pyrantel pamoate). The research protocol was approved by the University of Missouri Animal Care and Use Committee (No. 3791).
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In a 3 x 3 replicated Latin square design, nine yearlings were individually supplemented once daily with one of three treatments: 1) no FOS (CON); 2) 8 g of FOS/d (LOW); and 3) 24 g of FOS/d (HIGH). The FOS was 95% pure; two, three, or four residues long; and obtained from Golden Technologies Corp. (Golden, CO). The FOS was in dry powder form and was mixed with grain immediately before the morning feeding. The LOW treatment was extrapolated from doses given to dairy calves based on BW that elicited a response in fecal bacterial populations (our unpublished observations). The HIGH treatment was then added. The trial consisted of three consecutive 10-d feeding periods. The horses were blocked by BW into three groups of three horses, with one horse from each group receiving a different treatment during each 10-d treatment period, such that all horses received all treatments.
Sample Collection
Horses were given 7 d to acclimate to the diet and single fecal samples were collected from the surface of rubber-matted stall floors from each horse (to fill a 250-mL container) immediately after defecation during the 0730 feeding on the last 3 d of each 10-d feeding period. Horses routinely defecated within 2 h of being brought into stalls.
Fecal pH
Fecal pH was determined immediately after defecation by thoroughly mixing equal amounts of feces and double-deionized water in 50-mL tubes and submerging the pH probe (model 115, general purpose combination glass probe; Corning Science Products, Corning, NY) in the mixture until the reading stabilized. The pH meter was calibrated immediately before data collection.
Microbial and VFA Analyses
For later analysis of Lactobacilli, Escherichia coli, and Bifidobacteria populations, approximately 10 g of feces from the single fecal sample was mixed with 10 mL of an autoclaved glycerol salt solution (FDA, 1998
), placed on ice, and then frozen at 80°C until further processing. Fecal samples were thawed in an anaerobic chamber and serially diluted in anaerobic diluent (Bryant and Burkey, 1953
). Three dilutions were inoculated onto different plates to maximize counting precision. For Lactobacillus, samples were diluted from 104 to 106, and for Bifidobacteria and E. coli, samples were diluted from 102 to 104. The media for Lactobacillus bacteria was anaerobic de Man, Rogosa, and Sharpe (MRS) broth (#288130; Difco, Sparks, MD) and agar (20 g/L), with 20 mg/L vancomycin supplement and bromocresol green (LAMVAB; Hartemink et al., 1997
). The media for Bifidobacteria was a modified Columbia agar, containing 0.5% (vol/vol) propionic acid and adjusted to a pH of 5.0 (Beerens, 1990
). This medium is both elective and selective for Bifidobacteria. Confirmation of Bifidobacteria was by fructose-6-phosphate phosphoketolase test (Scardovi, 1986
). Incubation time for Lactobacilli and Bifidobacteria was 48 h at 37°C in an anaerobic gas chamber (Model 1025; Forma Scientific, Inc., Marietta, OH) under 85% N2, 10% H2, and 5% CO2. The E. coli was enumerated (cfu/mL) by a Petrifilm method according to the manufactures instruction (Petrifilm; 3M, St. Paul, MN). Fecal contents were measured for concentrations of acetate, butyrate, propionate, and lactate by gas chromatography (Varian Model 3400; Varian Instrument Group, Walnut Creek, CA; Goetsch and Galyean, 1983
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Fecal Consistency. Fecal consistency was recorded on collection days and subjectively scored on a scale from 1 to 5, with 1 being extremely dry, 3 being normal, and 5 being watery diarrhea. This scale was assigned by the authors, and fecal consistency was ranked by the same individual throughout the duration of the study.
Statistical Analyses. Data were analyzed by ANOVA using the PROC GLM procedures of SAS (SAS Inst., Inc., Cary, NC). The statistical model included the effects of treatment CON (0), LOW (8), and HIGH (24), period (I, II, or III), square, and horse within square. The effect of horse contained 8 df in the ANOVA. This 8 df came from the variation due to square (three Latin squares) plus horse within square. Linear and quadratic effects were tested in the form of contrasts for unequally spaced treatments. An alpha level of 0.05 was used for determination of statistical significance.
| Results |
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| Discussion |
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Excess dietary intake of carbohydrates by the horse results in starch fermentation in the hindgut, leading to increased lactate production and consequently low and potentially detrimental cecal pH values (Garner et al., 1978
; Bailey et al., 2002
). This may contribute to bouts of colic (Reeves et al., 1996
) and laminitis (Garner et al., 1978
; Bailey et al., 2002
) in the horse. In the current study, we did not collect cecal samples; therefore, we were unable to determine whether a correlation between cecal and fecal pH existed. A correlation between cecal and fecal pH has been reported in rats with fecal pH consistently being greater than cecal pH (Campbell et al., 1997
). In the present study, the decrease in fecal pH observed as a result of FOS supplementation caused no deleterious effects on any of the horses during the study or after its completion.
Fecal acetate, butyrate, propionate, lactate, and total fecal VFA concentrations all increased as the quanitity of FOS supplemented in the diet increased. This finding suggests that by providing FOS as an energy source for bacterial populations, VFA production increased, resulting in additional energy available for the horse. Our findings are in contrast with those of Campbell et al. (1997)
, who found no difference in fecal short-chain fatty acid concentrations of rats fed FOS vs. those fed the control diet. Campbell et al. (1997)
also found that cecal VFA concentrations were not correlated with fecal VFA concentrations; however, fecal VFA concentrations have served as indicators of fermentation patterns in the large intestine of dairy cows (Meylan et al., 2002
). Although fecal VFA concentrations may not represent actual cecal VFA concentrations, fecal VFA concentrations can provide relevant information regarding increases or decreases in VFA production, as was demonstrated in the present study.
It has been shown in humans, rats, and pigs that competitive exclusion of harmful bacteria by beneficial bacteria has the potential to improve intestinal heath (Gibson and Roberfroid, 1995
; Howard et al., 1995
; Campbell et al., 1997
). An increase in the population of pathogenic bacteria has been associated with enterocolitis, severe diarrhea, and equine dysautonomia (East et al., 2000
; Weese et al., 2001
; Garrett et al., 2002
). In the present study, the fecal population of Lactobacilli was not altered as a result of FOS supplementation, but the number of E. coli was decreased in horses supplemented with 8 g of FOS daily; however, fecal E. coli numbers for the HIGH horses did not differ from those of the control group. This finding may indicate that the optimal dose of FOS supplementation needed to decrease E. coli population in these yearlings was between 8 and 24 g. Campbell et al. (1997)
also reported no difference in fecal Lactobacilli population between control rats and those supplemented with FOS, but they reported an increased fecal Bifidobacteria population, as well as overall anaerobes. Bouhnik et al. (1999)
reported a significant correlation between the dose of FOS ingested and fecal Bifidobacteria counts in healthy humans. Increased population of Bifidobacteria are often reported in species supplemented with FOS or other oligosaccharides, as this species of bacteria is able to efficiently utilize FOS as an energy source (Howard et al., 1995
; Campbell et al., 1997
; Bouhnik et al., 1999
). Because we were unable to confirm the presence of Bifidobacteria in this study, we can neither refute nor substantiate the relationship of E. coli to Bifidobacteria. Additionally, because a limited number of species were measured, important information on fecal bacterial content is lacking. Research is needed to evaluate a broader array of microbial species to provide a more complete understanding of the fecal microbial environment and how it is affected by FOS supplementation. It also should be noted that fecal bacterial populations may not represent what has colonized the intestine, but rather what has passively moved through the intestine (Weese et al., 2003
). Although the result of the present study demonstrated an effect of FOS on fecal E. coli population, whether this is representative of cecal microbial populations requires further investigation.
| Implications |
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| Footnotes |
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2 The authors thank K. J. Willer for laboratory assistance and M. Ellersick for statistical support. ![]()
3 Correspondence: 111 Animal Sciences Research Center (phone: 573-882-0834; fax: 573-882-6827; e-mail:KerleyM{at}missouri.edu).
Received for publication July 28, 2004. Accepted for publication April 7, 2005.
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