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Characterization of microbial populations and volatile fatty acid concentrations in the jejunum, ileum, and cecum of pigs weaned at 17 vs 24 days of age

Department of Animal Science, University of Tennessee, Knoxville 37901-1071

² Correspondence:
313 Lenox Court, Springfield, TN 37172 (phone: 615-382-8297; fax: 615-384-1076; E-mail:
mfranklin{at}lallemand.com).

	Abstract

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In a series of five 17-d replicate trials, a total of 54 cannulated and 12 noncannulated pigs were used to determine the effects of weaning age (17 d or 24 d) on pH, dry matter percentage, aerobic and anaerobic microflora, lactate, and volatile fatty acid (VFA) concentrations in the jejunum, ileum, and cecum of weanling pigs. At ~14 d of age, cannulated pigs were surgically fitted with T-cannulas in the jejunum (n = 20), ileum (n = 18), or cecum (n = 16). Upon weaning, cannulated pigs were individually caged in an environmentally controlled room with ad libitum access to a phase starter diet and water. Noncannulated pigs were killed at weaning and samples were collected from the jejunum, ileum, and cecum. Digesta and fecal swabs from cannulated pigs were collected twice weekly. The pH of cecal contents was lower (P < 0.05) and dry matter percentage was greater (P < 0.05) than those of jejunal or ileal contents. Pigs weaned at 24 d of age had increased (P < 0.05) E. coli populations 3 d postweaning compared to preweaning populations, regardless of site of collection, whereas this increase was not observed in pigs weaned at 17 d of age. Unweaned pigs maintained higher (P < 0.05) lactobacilli populations compared to weaned pigs; however, populations declined (P < 0.05) in both groups by 3 d postweaning, with pigs weaned at 24 d of age having lactobacilli populations greater than pigs weaned at 17 d of age. Fecal populations of E. coli and lactobacilli declined (P < 0.05), whereas fecal bifidobacteria populations increased (P < 0.05) postweaning, regardless of weaning age. Concentrations of total fecal anaerobes declined (P < 0.05) in pigs weaned at 17 d of age but were maintained in pigs weaned at 24 d of age. Volatile fatty acid concentrations were greater (P < 0.05) in the cecum than in the jejunum or ileum, and acetic acid concentrations decreased (P < 0.05) postweaning regardless of weaning age. A tendency for L+ lactate concentrations to be greater (P < 0.07) in the ileum and jejunum vs the cecum was observed. Results indicate that weaning and weaning age have significant effects on microbial populations and VFA concentrations.

Key Words: Cecum • Ileum • Intestinal Microorganisms • Jejunum • Pigs • Weaning

	Introduction

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Intestinal microflora play a number of important roles in livestock production. Ultimately, these organisms can affect animal health, waste management, and even food-borne pathogens. Yet in pigs, characterization of the microflora and their by-products has been limited to only a few sites in the gastrointestinal tract, and information is still lacking with regard to the effects of dietary components, weaning, stressors, and therapeutics. As a result, swine research, particularly in the areas of young animal health, nutrient utilization, odor management, and food safety, has been hampered. As with the microflora, by-products of microbial activity, including volatile fatty acids (VFA), have been investigated by only a few researchers and in limited areas of the gastrointestinal tract in swine.

In pigs, changes in microfloral populations have been observed following weaning (Kenworthy and Crabb, 1963; Etheridge et al., 1984; Mathew et al., 1998). Other work has included effects of diet on VFA (Mathew et al., 1994, 1996). It is reasonable to believe that other factors may affect the microflora and their by-products, thus possibly providing an opportunity to manipulate these variables to increase productivity and decrease some detrimental aspects of livestock production.

The intestinal microflora of young pigs has been studied primarily through the sacrifice of littermates (Kenworthy and Crabb, 1963; Kuhn et al., 1993) and the use of ileal cannulas (Mathew et al., 1994, 1996). Variation in microfloral populations among pigs in the same litter also has been observed (Kenworthy and Crabb, 1963; Kuhn et al., 1993). While cannulation techniques have allowed for repeated samplings over the course of weaning or other transitional changes, most of these studies have focused on a single site, primarily the ileum. The objective of this study was to characterize microfloral and VFA concentrations in three sites of the gastrointestinal tract (including the jejunum, ileum, and cecum) of young swine during the weaning transition.

	Materials and Methods

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Experimental Design.

A total of 54 weanling pigs from nine litters were used in five replicate trials in this study. Pigs were from Yorkshire x Landrace sows bred to a Hampshire boar. At ~14 d of age, pigs were cannulated in the jejunum (n = 20), ileum (n = 18), or cecum (n = 16) in accordance with the Institutional Animal Care and Use Committee of The University of Tennessee, Knoxville. The surgical procedures for ileal and cecal cannulations have been previously described (Walker et al., 1986; Sutton et al., 1991). Jejunal cannulations were a modification of the ileal cannulation technique (Walker et al., 1986). Cannulas were placed in the jejunum on the cranial side of the terminal portion of the mesenteric lymph nodes. This corresponded to cannulas being approximately 30 cm from the ileocecal junction in pigs of this age. Cannulas were specially constructed (Mechanical Engineering Department, Purdue University, West Lafayette, IN) from Delrin 600 plastic.

Following surgery, pigs were returned to their respective sows and allowed a 3-d recovery period before initiation of the experiment. Pigs were then randomly assigned to a weaning age, with half of the pigs being weaned at 16 to 17 d of age and the other half remained on the sow until 23 to 24 d of age. At weaning, pigs were housed individually in stainless steel cages, provided approximately 1.9 m² in an environmentally controlled room, and were allowed ad libitum access to a starter diet and water. The diet was based on corn and soybean meal and contained 4% spray-dried plasma and 4% menhaden fishmeal. Orts and pig weights were taken to determine feed intake and growth rates.

Sampling.

Intestinal contents were collected at 17, 20, 24, 27, 31, and 34 d of age in both weaning groups, and collection was initiated at approximately 0700 each sampling day. At that time a rubber balloon with no head space was placed on the opened cannula and intestinal contents were allowed to collect until approximately 10 g of digesta had accumulated. Because digesta flow was intermittent, balloons were replaced approximately every 30 min until digesta began to flow consistently. Typically, once the digesta started flowing, approximately 15 min was needed for sufficient collection of material. Samples were immediately placed on ice until sample preparation began. This procedure was followed to minimize the possibility of bacterial proliferation and/or fermentation in the balloons. Fecal material swabs were taken when the intestinal sampling was complete.

In preparation for analysis, 1 g of digesta was weighed into a test tube and fecal swabs were weighed to determine the amount of sample collected. Fecal swabs were then placed in a test tube and 9 mL of prereduced PBS was added to the fecal and digesta test tubes. These initial mixtures were immediately placed in an anaerobic chamber to minimize exposure to oxygen for anaerobe enumeration, and the mixtures were then removed from the chamber for aerobic enumeration once serial dilutions for anaerobe enumeration were complete. The anaerobic chamber was constructed from 0.5-in Plexiglas and assembled by the Biological Services Facility at the University of Tennessee, Knoxville. Fecal and digesta tubes were agitated for 1 min and 10-fold dilutions were then made from both sets of tubes using prereduced PBS as the diluent. Collection and preparation were typically completed within 2 h of collection initiation. Sample pH was determined from the remaining digesta using a Corning #345 pH meter (Corning, New York, NY) with a high-performance glass electrode (cat. #476390). Digesta were then centrifuged at 27,000 x g at 4°C for 15 min. The resulting supernate was collected and stored at -80°C until VFA and lactate analyses were performed.

Microbial Analysis.

For anaerobic culture, 50 µL of each serial dilution was plated in duplicate onto prereduced media inside the anaerobic chamber. Conditions inside the chamber were maintained by using purchased (National Welders Supply, Knoxville, TN) premixed gas containing 90% nitrogen, 5% carbon dioxide, and 5% hydrogen. Additionally, conditions were constantly monitored by an oxygen analyzer (Coy Laboratory Products, Detroit, MI) and oxygen levels were maintained with palladium catalysts to 100 ppm or less. Inoculated Petri dishes were then placed in canning jars with tightened lids, and were subjected to a vacuum prior to sealing. Jars were then placed in convection incubators for appropriate culture conditions. For aerobe culture, serial dilutions were removed from the anaerobic chamber and Petri dishes were inoculated and placed directly into incubators. Four media were utilized in this study and included: 1) lactose MacConkey agar (Difco, Detroit, MI) for enumeration of E. coli, 2) rogosa medium (Difco) for enumeration of lactobacilli, 3) modified Columbia medium (Beerens, 1990) for enumeration of bifidobacteria, and 4) medium 10 (Caldwell and Bryant, 1966) for enumeration of total anaerobes. Modified Columbia media was used with a modified enzyme test (J. Patterson, personal communication) to detect the fructose-6-phosphate phosphoketolase enzyme characteristic to bifidobacteria. Escherichia coli were incubated aerobically at 37°C for 24 h. Anaerobic culture included bifidobacteria, which were incubated at 40°C for 48 h, lactobacilli, and total anaerobes, which were incubated at 37°C for 48 h. Bacteria were enumerated by visual count, disregarding atypical colonies. We confirmed our ability to select E. coli from other coliforms in swine intestinal samples through a preliminary study (Mathew et al., 1996). In that study, selected colonies were subjected to biochemical analysis (API 20, Vitek BioMerieux, Syosset, NY) to identify isolates to the species level. In all cases, colonies suspected of being E. coli (pink colonies with typical morphology on lactose MacConkey agar) were determined to be E. coli by biochemical analysis. The majority (96%) of isolates not phenotypical of E. coli were determined to be other enteric species, primarily Klebsiella spp. All large white colonies were counted as lactobacilli on Rogosa medium (Krause et al., 1994). Large white colonies were counted as bifidobacteria on modified Columbia media (Beerens, 1990) and all colonies were counted as total anaerobes on media 10 (Caldwell and Bryant, 1966).

VFA Analysis.

Volatile fatty acid concentrations were determined using a modified gas chromatographic method (Playne, 1985). Briefly, 1.5 mL of supernatant was mixed with 300 µL of 25% metaphosphoric acid (5:1 ratio) and incubated at room temperature for 30 min. Following centrifugation to remove the precipitate, 1 µL of sample was injected into a gas chromatograph (Model 5890, Hewlett Packard, Avondale, PA) with an HP-FFAP 10-m x 0.53-mm x 1-mm capillary column packed with cross-linked polyethylene glycol-TPA. A flame ionization detector was used with an oven temperature of 200°C, and a detector temperature of 250°C, for determination of acetate, propionate, butyrate, isobutyrate, valerate, and isovalerate concentrations. The lower detectable limit for all VFA was 0.1 mmol/L.

Lactate Analysis

Lactate concentrations were determined using a lactate analysis kit (#826-B; Sigma Chemical, St. Louis, MO). In that assay, 200 µL of the intestinal content supernatant was mixed with 400 µL of ice-cold 8% perchloric acid. Samples were incubated on ice for 30 min and centrifuged to remove precipitate. The resulting supernate was assayed according to Sigma, using the appropriate lactate dehydrogenase enzymes in separate assays for determination of D(-) and L(+) lactate. A modification of volumes was implemented to allow the use of microtiter plates and associated equipment. In the modified analysis, 200 µL of NAD–glycine solution was pipetted into a flat-bottom Serocluster 96-well microtiter plate (Costar Corp., Cambridge, MA). To that solution, 20 µL of each sample supernatant was added to triplicate wells. Absorbance was read at 340 nm on a microtiter plate reader (EL 340 Bio Kinetics, Bio-Tek Instruments, Wiooski, VT). Following the initial reading, 10 µL of the appropriate lactate dehydrogenase (L+ or D-) enzyme was added to each well. Plates were incubated for 15 min at 37°C followed by incubation for 15 min at room temperature. Following the final incubation, absorbance was read a second time. The lower detectable limit for the lactate analysis was 0.05 mmol/L.

Cortisol Analysis. Blood samples were obtained by vena cava puncture from both cannulated and noncannulated pigs, upon weaning and again at 1 wk postweaning. Samples were allowed to clot at 4°C for 1 h prior to centrifugation at 800 x g for 15 min. Serum was removed and stored at -20°C until cortisol analyses were performed. Serum concentrations of cortisol were determined using a standardized solid phase radioimmunoassay kit (Diagnostic Products Corporation, Los Angeles, CA). Twenty-five microliters of sample was used and sensitivity of the assay was 2 ng/mL. Coefficients of variation were less than 8% for all samples tested.

Statistical Analysis. The statistical model consisted of a randomized complete block design using repeated measures analysis with the individual pig serving as the experimental unit. Data were analyzed using the Mixed Model Procedure of SAS. Least squares means were separated using pairwise t-tests. Differences between days were determined using Pdmix procedures. Microbial and lactate concentrations were transformed (log₁₀) prior to statistical analysis.

	Results

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Pigs consumed 279 g/d of the starter diet and gained 144 g/d during this experiment. No differences were observed between weaning ages for intake or gain data. Cortisol concentrations did not differ (P > 0.05) between cannulated and noncannulated pigs, nor did cortisol levels differ (P > 0.05) in pigs weaned at 17 d of age (40 ng/mL) compared to pigs weaned at 24 d of age (56 ng/mL). Concentrations of E. coli in ileal contents were 7.8 and 7.7 log₁₀ cfu (P > 0.7) and those of lactobacilli were 9.3 and 9.1 log₁₀ cfu (P > 0.33) in noncannulated and cannulated pigs, respectively, when pigs were sacrificed at the end of the trial. No differences were noted in total anaerobes or bifidobacteria between noncannulated and cannulated pigs, with total anaerobe concentrations of 9.9 and 9.7 log₁₀ cfu (P > 0.11), and bifidobacteria concentrations of 6.7 and 6.7 log₁₀ cfu (P > 0.95) being observed in noncannulated and cannulated pigs, respectively. The pH did not differ between weaning groups, and thus Table 1 represents data pooled over weaning treatments. The pH of the cecal contents was lower (P < 0.05) than that of the ileal and jejunal contents, but did not differ (P > 0.05) between ileal or jejunal contents (Table 1).

In pigs weaned at 17 d of age, E. coli populations averaged for intestinal sites did not change (P > 0.05) 3 d postweaning, whereas populations increased (P < 0.05) 3 d postweaning in pigs weaned at 24 d of age (Table 2). There was a tendency (P < 0.08) for lactobacilli to be found in greater populations in the cecum (9.75 log₁₀ cfu/g) vs the ileum (9.08 log₁₀ cfu/g), with populations in the jejunum being intermediate.

Populations of E. coli and lactobacilli in fecal material declined (P < 0.05) after weaning regardless of weaning age; by contrast, fecal bifidobacteria concentrations increased (P < 0.05) after weaning regardless of weaning age (Table 3). Populations of total anaerobes found in feces were observed to decline (P < 0.05) in pigs weaned at 17 d of age, but were maintained in pigs weaned at 24 d of age (Table 3).

	Discussion

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No postweaning increases in E. coli were observed in pigs weaned at 17 d of age, in agreement with previous studies (Etheridge et al., 1984; Mathew et al., 1998), but in contrast with others (Mathew et al., 1993, 1996; McAllister et al., 1979), where E. coli populations increased after weaning. Mathew et al. (1998) postulated the absence of an E. coli increase may be due to weaning pigs into a highly sanitized, environmentally controlled room with limited contact among pigs. We also observed E. coli populations to be lower in pigs remaining on the sow, as have other investigators (Etheridge et al., 1984; Mathew et al., 1996). Lactobacilli populations declined postweaning in both groups of pigs, but populations fell to lower levels in pigs weaned at the earlier age compared to pigs weaned at the later age (Table 2). This may indicate that pigs weaned at older ages are more adept at dealing with changes over the course of weaning. Cranwell et al. (1976) found HCl production to be an age-related factor, with HCl production more stable in pigs at least 24 d of age. This may have direct implications to lactobacilli, which prefer lower pH (Kandler and Weiss, 1986). In another study, similar results were found with regard to pH and lactobacilli populations in pigs weaned at 21 d vs 28 d of age (Mathew et al., 1996). Fecal populations of lactobacilli and E. coli followed patterns typical of those observed in the more anterior portions of the gastrointestinal tract in this study. However, fecal bifidobacteria populations increased postweaning, possibly due to the decrease in lactobacilli and E. coli in the posterior gastrointestinal tract. The loss of direct competition may benefit other bacterial populations, including bifidobacteria. An increased fermentable substrate concentration may also have resulted from the decline in certain bacterial populations or from more substrate reaching the large intestine in pigs consuming weaning diets. Because anaerobes far outnumber aerobes in the gastrointestinal tract (McAllister et al., 1979), we looked at total anaerobic populations as an overall indicator of stability in the gastrointestinal tract. Total fecal anaerobe populations returned to preweaning levels by the end of the study in pigs weaned at 24 d of age, whereas populations in pigs weaned at 17 d decreased through the end of the study. No differences were observed in total anaerobes in cannulated sites of the gastrointestinal tract. Evidently, these changes are restricted to regions posterior to our cannula placement in this study. These data also seem to indicate total anaerobes recover more quickly from weaning stresses in pigs weaned at later ages than pigs weaned at younger ages.

Cecal concentrations of VFA indicate more fermentation occurs in this region than in the ileum or jejunum. The ileal, jejunal, and cecal concentrations of VFA found in this study agree with those from another study in our laboratory (Mathew et al., 1998); however, cecal and jejunal VFA levels were only obtained by killing animals at the end of that experiment. Thus, direct comparisons of these studies over the course of the weaning were not possible.

The increased VFA concentrations coincide with increased microbial populations found in the cecum, thus indicating greater microbial fermentation. Elsden et al. (1946) demonstrated that VFA production follows bacterial populations, and Imoto and Namioka (1978) showed the major site of VFA production in the pig to be the large intestine. Changes in fermentation patterns postweaning are also indicated by shifts in lactate production (Mathew et al., 1998), as was observed in this study for the L(+) isomer, which increased after weaning. Pigs in this study demonstrated normal daily intake and appeared to grow at a rate typical of pigs of this age (NRC, 1998). This study indicates VFA declines post weaning, which may have significant effects on the energy available to the intestinal mass. Large intestinal VFA production in the pig has been estimated to contribute between 5 and 28% of the total maintenance energy requirement (Friend et al., 1964; Imoto and Namioka, 1978). In other species, VFA have been shown to be the primary energy source to the intestinal mass, contributing up to 70% of the required maintenance needs (Imoto and Namioka, 1978). Because of the importance of the microflora and their activities and by-products, further research is warranted to determine optimal conditions to maintain the gastrointestinal health of the weaned pig.

	Implications

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Because of the overall importance of intestinal microflora to animal health, nutrient and waste management, and food safety, it is important that these organisms and their by-products continue to be characterized and that methods for such investigations be validated. The ability to cannulate several regions of the gastrointestinal tract without affecting the microflora was demonstrated in this study, thus increasing confidence that this method can be used to study effects of diet, treatments, and stressors on the microflora. This research also demonstrated that weaning can affect microbial populations in various regions of the gastrointestinal tract, as we were able to characterize changes directly associated with weaning in several bacterial groups across various regions of the gastronintestinal tract and noted that bacterial by-products followed similar patterns. Such information should provide additional insight into the complicated interplay of intestinal microflora and the host animal.

	Footnotes

 
¹ Present address: Lallemand Animal Nutrition, Inc.

Present address: The Iams Company, Lewisburg, OH 45338-0189.

Received for publication July 3, 2001. Accepted for publication June 26, 2002.

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