Poult. Sci.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Poult Sci 2008. 87:1263-1275. doi:10.3382/ps.2007-00179
© 2008 Poultry Science Association
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Donalson, L. M.
Right arrow Articles by Ricke, S. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Donalson, L. M.
Right arrow Articles by Ricke, S. C.

ENVIRONMENT, WELL-BEING, AND BEHAVIOR

In Vitro Fermentation Response of Laying Hen Cecal Bacteria to Combinations of Fructooligosaccharide Prebiotics with Alfalfa or a Layer Ration

L. M. Donalson*,1, W. K. Kim*,2, V. I. Chalova*,3, P. Herrera*,3, J. L. McReynolds{dagger},4, V. G. Gotcheva{ddagger}, D. Vidanovic§, C. L. Woodward*, L. F. Kubena{dagger}, D. J. Nisbet{dagger} and S. C. Ricke*,3

* Department of Poultry Science, Texas A&M University, College Station 77843-2472; {dagger} USDA-ARS, Southern Plains Agricultural Research Center, Food and Feed Safety Research Unit, College Station, TX 77845; {ddagger} Department of Biotechnology, University of Food Technologies, Plovdiv 4002, Bulgaria; and § Department of Food Hygiene and Technology, Faculty of Veterinary Medicine, Belgrade University, 11000, Serbia

4 Corresponding author: mcreynolds{at}ffsru.usda.gov


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
The objective of this in vitro study was to evaluate the effects of combining a prebiotic with alfalfa on fermentation by laying hen cecal bacteria. Cecal contents from laying hens were diluted to a 1:3,000 concentration with an anaerobic dilution solution and added to serum tubes filled with ground alfalfa or a layer ration with or without fructooligosaccharide (FOS) prebiotic. Samples were processed in an anaerobic hood, pressurized by using a pressure manifold, and incubated at 37°C. Volatile fatty acid (VFA) and lactic acid concentrations were quantified at 6 and 24 h of substrate fermentation. In this study, fermentation of alfalfa resulted in greater production of acetate, VFA, and lactic acid compared with the layer ration. Although with a relative inconsistency in data between trials, the amendment of FOS to both alfalfa and the layer ration appeared to further increase fermentation as demonstrated by overall higher propionate, butyrate, VFA, and lactic acid concentrations. The effect was more pronounced after 24 h of fermentation, implying time constraints for the optimal production of fermentation products in the chicken gastrointestinal tract. These data indicate that in vitro cecal fermentation can be enhanced by the addition of FOS.

Key Words: alfalfa • fermentation • fructooligosaccharide • laying hen


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Feeding laying hens high fiber sources such as alfalfa has been suggested as an alternative to the conventional molting method of feed withdrawal (Donalson et al., 2005; Landers et al., 2005a,b; Woodward et al., 2005; McReynolds et al., 2006). In addition to addressing physiological and food safety issues, alfalfa meal diets have been shown to be fermentable by cecal microflora when incubated in vitro (McReynolds et al., 2005, 2006; Woodward et al., 2005; Saengkerdsub et al., 2006; Dunkley et al., 2007; Landers et al., 2007). Alfalfa is a readily available, high-protein, high-fiber feedstuff with one of the slower rates of passage through the avian gastrointestinal system (Hanson, 1972; Sibbald, 1979; García et al., 2000).

The majority of fermentation in laying hens occurs in the ceca, which provides a stable environment for indigenous microflora such as Bifidobacterium, Eubacterium, and Propionibacterium (Guo et al., 2003; Józefiak et al., 2004). The microflora ferment undigested dietary compounds such as plant polysaccharides to produce short-chain fatty acids (SCFA) or volatile fatty acids (VFA), ammonia, carbon dioxide, methane, and hydrogen (Tsukahara and Ushida, 2000). Short-chain fatty acids such as acetate, propionate, and butyrate have nutritional value to the animal because they provide energy for the hen that would otherwise not be utilized in the absence of microbial fermentation. Tsukahara and Ushida (2000) estimated that 30 to 40% of the maintenance energy for monogastrics is derived from microbial fermentation.

The addition of prebiotics to diets has been shown to increase fermentation both in vitro (Rycroft et al., 2001) and in vivo (Xu et al., 2003). Prebiotics were defined by Gibson and Roberfroid (1995) as indigestible food ingredients that are capable of stimulating the growth of selective bacteria and benefiting the host. Recent concerns about antibiotic resistance have forced producers in Europe to discontinue the use of antibiotics, and there is a potential for the same trend in the United States (Jones and Ricke, 2003; Patterson and Burkholder, 2003). Prebiotics have been shown to reduce pathogen colonization, alter the microbial community, prevent cancer (in mammals), and reduce cholesterol (Patterson and Burkholder, 2003). Fructooligosaccharide (FOS) is a naturally occurring oligosaccharide, usually of plant origin, and is the only product recognized and used as a food ingredient and prebiotic (Gibson and Roberfroid, 1995; Bomba et al., 2002). Because of the β-linkages possessed by FOS, it is able to resist enzymatic degradation and absorption in the upper gastrointestinal tract to reach the cecum, where the majority of fermentation occurs in chickens (Gibson and Roberfroid, 1995; Xu et al., 2003; Józefiak et al., 2004; Juskiewicz et al., 2004).

In poultry, oligosaccharides reach the hindgut and alter lower intestinal tract physiology and function, which could be beneficial in preventing bacterial contamination on broiler carcasses and in eggs (Orban et al., 1997). Fermentation from prebiotics includes shifts in the production of end products such as hydrogen, carbon dioxide, bacterial cell mass, and, most important, SCFA (Cummings et al., 2001). Short-chain fatty acids have been shown to increase the absorption of calcium, magnesium, and iron (Gibson and Roberfroid, 1995) and to modify the bacterial ecosystem in the ceca. The objective of this study was to evaluate the effects of combining a prebiotic (FOS) with alfalfa on the in vitro production of fermentation acids from laying hen cecal bacteria.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Animals and Cecal Preparation
Laying hens aged between 100 to 105 wk old were obtained from a commercial laying facility. Birds were housed 1 per cage at the Texas A&M University Poultry Science Research Center in College Station, Texas, and allowed to acclimate for a minimum of 1 wk. During this time, the birds were fed a complete layer ration (Table 1Go) ad libitum and allowed full access to water. All animal handling procedures were approved by the Texas A&M University Institutional Animal Care and Use Committee. Three hens were chosen at random, exsanguinated, and terminated using CO2 asphyxiation. The cecal contents from the birds were squeezed out into an empty sterile 30 x 115 mm, 50-mL conical tube (Becton, Dickinson and Company, Franklin Lakes, NJ) and mixed thoroughly to obtain a uniform pooled sample. Approximately 0.1 g of the cecal pool was subsequently diluted to a 1:3,000 concentration (wt/vol) with anaerobic phosphate buffer in an anaerobic chamber (10% CO2, 5% H2, and 85% N2 gas phase; Coy Laboratory Products, Ann Arbor, MI). The 1:3,000 dilution was considered the most suitable for this study based on preliminary experiments (data not shown) that had examined several dilutions to obtain VFA and lactic acid concentrations within the range of detection of the gas chromatograph. Anaerobic phosphate buffer was prepared as described previously (Bryant and Robinson, 1961), with the addition of cysteine-HCl before autoclaving (Shermer et al. 1998).


View this table:
[in this window]
[in a new window]

  		Table 1. Composition of layer ration and alfalfa-fructooligosaccharide (FOS) substrates for in vitro fermentation
 
Procedure
Two substrates, alfalfa meal (A) and layer ration (LR) with and without FOS (Encore Technologies, Plymouth, MN), were examined for fermentation properties in 2 trials. Alfalfa meal was obtained from a local cooperative, whereas the LR (Table 1Go) was obtained from the Texas A& M University Poultry Science Center feed mill in College Station, Texas. Approximately 0.25 g of either substrate and 7.5% FOS were added where appropriate to presterilized 20-mL serum tubes. Aliquots of 5 mL of diluted, pooled cecal contents were added to the tubes under anaerobic conditions. A tube with only cecal inoculum (I) was used as the control. Fermentation was conducted at 37°C for 0, 6, or 24 h. At each time point, a 2-mL aliquot was obtained for VFA and lactic acid concentration analysis. Final concentrations of VFA and lactic acid were determined by subtracting the respective values of baseline fermentation products at time 0 h from values at times 6 and 24 h.

Cecal VFA and Lactic Acid Concentrations
Volatile fatty acid concentrations (acetic, propionic, butyric, isobutryic, valeric, and isovaleric acids) were determined by gas-liquid chromatography as described by Corrier et al. (1990). The analysis was conducted with a gas chromatograph equipped with a flame-ionization detector and a peak profile integration-quantification integrator (model 110 gas chromatograph, SR1 Instruments, Torrence, CA). Each sample peak profile was integrated and quantified relative to an internal standard of methylbutyric acid placed in the same sample. Lactic acid concentrations were determined by an enzymatic method (Hohorst, 1974).

Statistical Analysis
Data were analyzed by using the GLM procedure of SAS (2001, SAS Institute Inc., Cary, NC). Differences among treatment groups (I only; I and FOS; A and I; A, FOS, and I; LR and I; LR, FOS, and I) were determined by 1-way ANOVA, and means were separated by using Duncan’s multiple range test. Treatments were performed in triplicate in each trial. All results were considered significant at P ≤ 0.05.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS
 REFERENCES
 
At h 6, A, A and FOS (AF), LR, and LR and FOS (LRF) in both trials exhibited higher (P ≤ 0.05) acetic acid concentrations (Figure 1Go) than I. At 24 h, the trend was similar, showing that fermentation had occurred after 6 h. At 24 h in both trials, the A and AF treatments continued to yield the greatest increases in acetic acid concentrations. The current results agree with the concept of acetate being a predominant end product of a successful fermentation of alfalfa silage (Owens et al., 2002). Kass et al. (1980) demonstrated that the addition of 40% alfalfa to the regular diets of growing swine caused an increase in acetate production in both the cecum and colon. The structural composition of alfalfa may contribute to the greater increases in acetic acid when compared with LR treatments because of the nearly 10-fold difference in cellulose concentration between alfalfa and corn (Church, 1977). Acetic acid is a primary VFA produced from cellulose fermentation by numerous anaerobes (Van Soest, 1982). Salanitro et al. (1978) isolated anaerobic bacteria from chicken intestines capable of constant synthesis of large amounts of acetate (41 to 50 µmol/mL). Using a nonselective DNA method, Apajalahti et al. (2001) determined gastrointestinal microbial profiles of broiler chickens from 8 commercial farms and reported Bacteroides, Clostridium, Fusobacteria, and Bifidobacterium to be the second most abundant genera. The same genera appeared to be involved in avian cecal fermentation and particularly in acetate production (Józefiak et al., 2004). When Dunkley et al. (2007) incubated various high-fiber feed substrates with cecal inocula in vitro for 24 h, acetate was generally the primary SCFA, followed by propionate and butyrate. However, when compared with soybean meal, significant differences in acetate from alfalfa or other high-fiber substrates after fermentation were not always observed. Saengkerdsub et al. (2006) also identified acetate as a primary fermentation product of alfalfa after being incubated in vitro with a chicken cecal bacterial mixture for 24 h, but no differences were observed in acetate quantities when compared with layer ration fermentation with or without methanogen inhibitors. When Woodward et al. (2005) compared in vivo acetic acid concentrations from hens fed alfalfa molt diets, a nonmolt layer ration, and feed-withdrawal molt diets, hens fed alfalfa yielded significantly lower acetic acid concentrations than birds fed the layer ration. The difference in acetic acid concentrations in our in vitro study and the in vivo trial by Woodward et al. (2005) may possibly be due to much lower feed intake of alfalfa (approximately 4- to 5-fold) when compared with fully fed hens. In addition, transit time in the ceca may have been shorter than the total time used for incubation in the in vitro studies. The present study used equal amounts of feed substrates and the cecal contents were consistent throughout all treatments, which would not be the case in vivo.


Figure 1
View larger version (29K):
[in this window]
[in a new window]

  		Figure 1. Increase in acetic acid concentration (µmol/mL) over the baseline after 6 and 24 h of fermentation. Numbers above equal concentrations at 6 and 24 h minus the baseline (time 0). Baseline values for trial 1 for the respective treatments: 4.63, 5.00, 5.23, 9.10, 5.73, and 3.97 µmol/mL. Baseline values for trial 2 for the respective treatments: 5.03, 5.37, 8.17, 9.27, 5.27, and 6.93 µmol/mL. Standard error bars are based on the average of 3 tubes per trial. I = inoculum only; IF = I + fructooligosaccharide (FOS); A = alfalfa; AF = A + FOS; LR = layer ration; LRF = LR + FOS. A,BMeans within trial 1 (striped bars) without a common letter differ (P ≤ 0.05). a–cMeans within trial 2 (white bars) without a common letter differ (P ≤ 0.05).

 
After 6 h of fermentation, propionic acid concentrations were higher (P ≤ 0.05) in the LR and LRF treatments than in all other treatments (trial 1). This same pattern was exhibited in trial 2, with LRF having higher (P ≤ 0.05) propionic acid concentrations than all treatments except A (Figure 2Go). These results confirmed the trend reported previously by Dunkley et al. (2007) for soybean cecal fermentation exhibiting highest level of propionate concentrations compared with alfalfa and alfalfa combinations with different levels of layer rations. Higher propionate concentrations in the cecum of growing swine fed a regular diet not containing alfalfa were observed by Kass et al. (1980) as well. In our study, however, after 24 h of incubations, AF yielded higher (P ≤ 0.05) propionic concentrations than all other treatments in both trials. This is in contrast with the results reported by Saengkerdsub et al. (2006), who did not observe any differences in the production of propionate from alfalfa and layer feed when fermented by chicken cecal microflora for 24 h with or without methanogen inhibitors.


Figure 2
View larger version (28K):
[in this window]
[in a new window]

  		Figure 2. Increase in propionic acid concentration (µmol/mL) over the baseline after 6 and 24 h of fermentation. Numbers above equal concentrations at 6 and 24 h minus the baseline (time 0). Baseline values for trial 1 for the respective treatments: 0.00 µmol/mL for all treatments. Baseline values for trial 2 for the respective treatments: 0.00 µmol/mL for all treatments. Standard error bars are based on the average of 3 tubes per trial. I = inoculum only; IF = I + fructooligosaccharide (FOS); A = alfalfa; AF = A + FOS; LR = layer ration; LRF = LR + FOS; ND = not detected. A–DMeans within trial 1 (striped bars) without a common letter differ (P ≤ 0.05). a–cMeans within trial 2 (white bars) without a common letter differ (P ≤ 0.05).

 
In both trials, all substrate treatments had significantly higher isobutyric acid concentrations than both the I and I and FOS (IF) treatments at 6 h (Figure 3Go). After 24 h, the general trend was similar, with the I and IF treatments producing significantly less isobutyric acid. Although hens in the fully fed (layer ration) treatment of Woodward et al. (2005) yielded higher isobutyric concentrations, they were not significantly different from the hens treated with alfalfa, which coincides with the results in the current trial after 6 and 24 h of fermentation.


Figure 3
View larger version (29K):
[in this window]
[in a new window]

  		Figure 3. Increase in isobutyric acid concentration (µmol/mL) over the baseline after 6 and 24 h of fermentation. Numbers above equal concentrations at 6 and 24 h minus the baseline (time 0). Baseline values for trial 1 for the respective treatments: 0.00, 3.37, 0.00, 3.26, 0.00, and 2.23 µmol/mL. Baseline values for trial 2 for the respective treatments: 0.00, 3.37, 0.00, 0.00, 0.00, and 3.30 µmol/mL. Standard error bars are based on the average of 3 tubes per trial. I = inoculum only; IF = I + fructooligosaccharide (FOS); A = alfalfa; AF = A + FOS; LR = layer ration; LRF = LR + FOS; ND = not detected. A,BMeans within trial 1 (striped bars) without a common letter differ (P ≤ 0.05). a–dMeans within trial 2 (white bars) without a common letter differ (P ≤ 0.05).

 
Butyric acid concentrations were variable in trial 1, and no differences (P > 0.05) were apparent between treatments in trial 2 after 6 h (Figure 4Go). Similarly, no diet effect on butyric acid production was reported by Moore et al. (2004), who did not observe differences among all treatments (fully fed and feed withdrawal and zinc-containing diets) in 2 of 3 in vivo trials. It also appears that the increase in the dietary fiber concentrations in swine diets in the form of alfalfa does not increase butyric acid concentrations in the swine cecum (Kass et al., 1980). In our study, however, after 24 h of fermentation, butyric acid concentration levels increased markedly, with AF being significantly higher than all other treatments in trial 1 and being significantly higher than both the I and A treatments in trial 2. The highest in vitro concentrations of butyrate were found in some trials by Dunkley et al. (2007) after fermenting feed containing 90 or 70% alfalfa for 24 h in cecal inocula. Saengkerdsub et al. (2006), however, reported that layer feed fermentations with chicken cecal inocula yielded approximately 2-fold higher levels of butyric acid concentrations compared with alfalfa fermentation.


Figure 4
View larger version (29K):
[in this window]
[in a new window]

  		Figure 4. Increase in butyric acid concentration (µmol/mL) over the baseline after 6 and 24 h of fermentation. Numbers above equal concentrations at 6 and 24 h minus the baseline (time 0). Baseline values for trial 1 for the respective treatments: 1.06, 3.32, 3.36, 3.33, 2.14, and 3.27 µmol/mL. Baseline values for trial 2 for the respective treatments: 0.00, 3.67, 2.17, 3.23, 1.10, and 3.27 µmol/mL. Standard error bars are based on the average of 3 tubes per trial. I = inoculum only; IF = I + fructooligosaccharide (FOS); A = alfalfa; AF = A + FOS; LR = layer ration; LRF = LR + FOS. ACMeans within trial 1 (striped bars) without a common letter differ (P ≤ 0.05). a,bMeans within trial 2 (white bars) without a common letter differ (P ≤ 0.05).

 
After 6 h of fermentation in trial 1, isovaleric acid concentrations in the AF treatment were significantly higher than in all other treatments except LR and LRF (Figure 5Go). Similarly, in trial 2, AF continued to produce more isovaleric acid, but these levels were different (P ≤ 0.05) only from I and IF. After 24 h of fermentation, the concentration of isovaleric acid varied among trials. Although no differences in isovalerate concentrations (P > 0.05) were observed in trial 1, significantly more production from the AF, LR, and LRF treatments was seen in trial 2. These results are comparable to the in vivo responses observed by Woodward et al. (2005), who reported that 2 out of the 4 trials exhibited no statistical differences between fully fed (layer ration) and alfalfa-fed hens. Donalson et al. (2007) did not observe any differences in isovaleric acid production in hens fed alfalfa meal or a layer ration. Under in vitro conditions, however, Dunkley et al. (2007) did not detect isovalerate as a fermentation product of any of the incubated feeds containing alfalfa.


Figure 5
View larger version (27K):
[in this window]
[in a new window]

  		Figure 5. Increase in isovaleric acid concentration (µmol/mL) over the baseline after 6 and 24 h of fermentation. Numbers above equal concentrations at 6 and 24 h minus the baseline (time 0). Baseline values for trial 1 for the respective treatments: 0.00 µmol/mL for all treatments. Baseline values for trial 2 for the respective treatments: 0.00, 0.90, 0.00, 0.00, 0.00, and 0.00 µmol/mL. Standard error bars are based on the average of 3 tubes per trial. I = inoculum only; IF = I + fructooligosaccharide (FOS); A = alfalfa; AF = A + FOS; LR = layer ration; LRF = LR + FOS. A,BMeans within trial 1 (striped bars) without a common letter differ (P ≤ 0.05). a–cMeans within trial 2 (white bars) without a common letter differ (P ≤ 0.05).

 
After 6 h of fermentation, valeric acid in the I, IF, A, and LR treatments could not be detected in trial 1 and in any treatments except LRF in trial 2 (Figure 6aGo). The results after 6 h of fermentation correspond to previous reports by Woodward et al. (2005) of nondetectable valeric acid in some trials. After 24 h of fermentation (Figure 6bGo), valeric acid could not be detected in either the IF or I treatments in trial 1. In trial 2, valeric acid concentrations were higher (P ≤ 0.05) in the A, AF, LR, and LRF treatments, with no differences (P > 0.05) observed between A and IF. The fermentation of A, AF, LR, and LRF resulted in valeric acid concentrations similar to those reported by Dunkley et al. (2007). According to their study, the in vitro fermentation of all combinations of layer rations and alfalfa produced valeric acid ranging from approximately 3 to 4 µmol/mL.


Figure 6
View larger version (28K):
[in this window]
[in a new window]

  		Figure 6. Increase in valeric acid concentration (µmol/mL) over the baseline after 6 and 24 h of fermentation. Numbers above equal concentrations at 6 and 24 h minus the baseline (time 0). Baseline values for trial 1 for the respective treatments: 0.00 µmol/mL for all treatments. Baseline values for trial 2 for the respective treatments: 0.00 µmol/mL for all treatments. Standard error bars are based on the average of 3 tubes per trial. I = inoculum only; IF = I + fructooligosaccharide (FOS); A = alfalfa; AF = A + FOS; LR = layer ration; LRF = LR + FOS; ND = not detected. A–CMeans within trial 1 (striped bars) without a common letter differ (P ≤ 0.05). a,bMeans within trial 2 (white bars) without a common letter differ (P ≤ 0.05).

 
When total VFA concentrations were evaluated after 6 h of fermentation, the A, AF, LR, and LRF treatments yielded higher (P ≤ 0.05) concentrations than the I and IF incubations (Figure 7aGo). After 24 h of fermentation, the AF treatment yielded higher (P ≤ 0.05) total VFA production than did all other treatments (Figure 7bGo). These results indicated that fermentation occurred in all incubated substrate combinations; however, the greatest amount of total VFA production occurred when A and FOS were both present as substrates. Overall, AF treatments yielded higher VFA concentrations than all other treatments. The results of this study did not correspond to those seen by Woodward et al. (2005) in vivo, in which fully fed (layer ration) hens had significantly higher total VFA concentrations than alfalfa-treated hens. Again, this may be due to the decreased feed intake of alfalfa-treated hens compared with fully fed hens observed in their study.


Figure 7
View larger version (33K):
[in this window]
[in a new window]

  		Figure 7. Increase in total volatile fatty acid (VFA) concentration (µmol/mL) over the baseline after 6 and 24 h of fermentation. Numbers above equal concentrations at 6 and 24 h minus the baseline (time 0). Baseline values for trial 1 for the respective treatments: 5.69, 11.69, 8.59, 15.69, 7.78, and 9.47 µmol/mL. Baseline values for trial 2 for the respective treatments: 5.03, 13.27, 10.34, 12.5, 6.37, and 12.4 µmol/mL. Standard error bars are based on the average of 3 tubes per trial. I = inoculum only; IF = I + fructooligosaccharide (FOS); A = alfalfa; AF = A + FOS; LR = layer ration; LRF = LR + FOS. A–CMeans within trial 1 (striped bars) without a common letter differ (P ≤ 0.05). a–d Means within trial 2 (white bars) without a common letter differ (P ≤ 0.05).

 
Lactic acid concentrations of AF in trial 1 after 6 h of fermentation yielded significantly higher values than did LR and LRF, whereas in trial 2, no significant differences were observed among the A, AF, LR, and LRF treatments (Figure 8aGo). At 24 h, results varied between trials; however, the trends were similar, with AF exhibiting greater lactic acid concentrations than LR in both trials (Figure 8bGo). Woodward et al. (2005) reported significantly greater lactic acid concentrations in ceca of alfalfa-fed hens compared with hens that were subjected to feed withdrawal. Likewise, Donalson et al. (2007) reported that in half of the trials, alfalfa- and alfalfa and FOS-treated hens yielded greater concentrations of lactic acid compared with hens fed a layer ration diet. Lactic acid is the primary fermentation product of Lactobacillus spp., which are considered to be beneficial bacteria in the gut. Fructooligosaccharide bypasses degradation in the upper gastrointestinal tract and stimulates the production of lactic acid by hindgut microflora such as Bifidobacterium (Tsukahara and Ushida, 2000), which is known to inhibit the growth of enteric pathogens (Fernandez et al., 2002).


Figure 8
View larger version (22K):
[in this window]
[in a new window]

  		Figure 8. Increase in lactic acid concentration (µmol/mL) over the baseline after 6 and 24 h fermentation. Numbers above equal concentrations at 6 and 24 h minus the baseline (time 0). Baseline values for trial 1 for the respective treatments: 0.00, 0.10, 2.40, 1.70, 0.47, and 0.13 µmol/mL. Baseline values for trial 2 for the respective treatments: 0.30, 0.70, 1.53, 2.50, 0.20, and 0.37 µmol/mL. Standard error bars are based on the average of 3 tubes per trial. I = inoculum only; IF = I + fructooligosaccharide (FOS); A = alfalfa; AF = A + FOS; LR = layer ration; LRF = LR + FOS. ACMeans within trial 1 (striped bars) without a common letter differ (P ≤ 0.05). a–cMeans within trial 2 (white bars) without a common letter differ (P ≤ 0.05).

 
When correlations among fermentation acids were evaluated (Table 2Go), high correlations were seen among individual VFA and total VFA (P < 0.0001). The overall trend was a high correlation between total VFA and propionic acid (0.9936) or acetic acid (0.9899). This is in agreement with the observations of Van der Weilen (2000), who showed that propionate and acetate were the dominant VFA produced in the ceca of chickens. An increase in propionic acid production has been used as an indicator of successful propiogenic establishment in the chicken ceca of probiotic cultures capable of exhibiting competitive exclusion against foodborne Salmonella colonization (Nisbet et al., 1996). The lowest correlation was seen between VFA and lactic acid (0.757). These results are consistent with those obtained by Van der Weilen (2000), who found that the increases in total VFA production were associated with the increases in acetate, propionate, and butyrate in the ceca of broiler chickens. The same study also showed a lack of correlation between lactobacilli numbers and total VFA concentrations.


View this table:
[in this window]
[in a new window]

  		Table 2. Statistical data for the correlations among various volatile fatty acid (VFA) and lactic acid concentrations from laying hen cecal bacteria
 
The rationale behind the current findings is that plant protein diets such as alfalfa are highly fermentable and may naturally lead to higher VFA production (Hanson, 1972; Tsukahara and Ushida, 2000). In our study, the inclusion of FOS appeared to further increase fermentation as shown by overall higher VFA and lactic acid concentrations. The effect was more pronounced after 24 h of fermentation, resulting in distinctive increases in the production of total VFA and lactic acid from substrates combined with FOS. Rycroft et al. (2001) also evaluated the time effects on fermentation of oligosaccharides and reported similar results. Although increases in VFA production at 6 h were demonstrated in the present study and at 5 h by Rycroft et al. (2001), large quantitative increases in VFA were observed only after 24 h of fermentation in both studies. Because the retention time of alfalfa in the gastrointestinal tract of force-fed roosters is close to 24 h (Sibbald, 1979, 1980) and the retention time of layer rations is even shorter, it would be less useful to examine the fermentation effects beyond 24 h.

Prebiotics have also been proven not to serve as carbon sources for enterobacteria such as Salmonella and Escherichia coli (Bailey et al., 1991; Xu et al., 2003). Although FOS can inhibit enteric pathogens and modify the metabolic activity of the normal microflora, it does not negatively influence the indigenous bacteria. An increase in VFA concentrations has been shown to have long-term beneficial effects on host health by providing increased energy (Guo et al., 2003) and by reducing Salmonella Enteritidis colonization (Ricke, 2003; Moore et al., 2004; Ricke et al., 2004; Woodward et al., 2005) and Enterobacteriaceae numbers in the ceca (Van der Wielen, 2000). In addition, Van der Wielen et al. (2000) suggested that VFA such as acetate, propionate, and butyrate aid in the development of the microflora in chickens.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
REFERENCES
 
The majority of fermentation occurs in the ceca of the chicken, which harbors indigenous microflora such as Bifidobacterium and Lactobacillus (Salanitro et al., 1974). When supplied with fermentable substrates, these bacteria benefit from the fermentation by-products such as VFA. In this study, fermentation was increased when alfalfa was provided as the feed substrate. Because of the high retention time of alfalfa as a primary dietary component, it could affect fermentation because of a longer exposure time when compared with cereal-based layer ration diets, which have a much shorter passage rate. Longer passage rates equal greater retention time, which should retard gut emptying and potentially serve as a barrier to pathogen colonization (Holt, 2003). The addition of FOS to an A diet enhanced fermentation as indicated by the increase of VFA and lactic acid production. Increases in lactic acid have been related to decreases in pH, thus inhibiting Salmonella crop colonization (Durant et al. 1999). In this study, FOS increased fermentation when combined with both A or an LR, with the maximum effect achieved after 24 h of incubations. Therefore, the longer passage rate and retention of diets appears to be desirable for maximizing fermentation. Although bacterial fermentation after 24 h could still be studied, the data generated would have limited value because most feed substrate passage rates in vivo are less than 24 h. In vivo studies will also help to determine the optimal levels of prebiotics that should be included in a poultry diet and the more specific effects that FOS has on particular functional groups of indigenous cecal microflora and pathogen colonization in chickens.


   ACKNOWLEDGMENTS
 
This research is supported by Hatch grant H8311 administered by the Texas Agricultural Experiment Station, USDA-NRI grant number 2002-02614 and U.S. Poultry and Egg Association grant #485. We would also like to thank Encore Technologies, Plymouth, MN for providing the FOS. LMD was partially supported by a Maurice Stein Fellowship, Poultry Science Association (Savoy, IL). P. H. was supported by USDA-NRI grant number 2005-35201-15429.


   FOOTNOTES
 
1 Current address: Southwest Foundation for Biomedical Research, San Antonio, TX 78245. Back

2 Current address: Department of Cardiology, David Geffen School of Medicine, Los Angeles, CA 90095. Back

3 Current address: Center for Food Safety, IFSE, and Department of Food Science, University of Arkansas, Fayetteville, AR 72704. Back

Received for publication May 2, 2007. Accepted for publication February 8, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Apajalahti, J. H. A., A. Kettunen, M. R. Bedford, and W. E. Holben. 2001. Percent G+C profiling accurately reveals diet-related differences in the gastrointestinal microbial community of broiler chickens. Appl. Environ. Microbiol. 67:5656–5667.[Abstract/Free Full Text]

Bailey, J. S., L. C. Blankenship, and N. A. Cox. 1991. Effect of fructooligosaccharide on Salmonella colonization of the chicken intestine. Poult. Sci. 70:2433–2438.[Web of Science][Medline]

Bomba, A., R. Nemcová, S. Gancarcikovc, R. Herich, P. Guba, and D. Mudronová. 2002. Improvement of the probiotic effect of micro-organisms by their combination with maltodextrins, fructooligosaccharides and polyunsaturated fatty acids. Br. J. Nutr. 88:S95–S99.[CrossRef][Web of Science][Medline]

Bryant, M. P., and I. M. Robinson. 1961. An improved nonselective culture medium for ruminal bacteria and its use in determining the diurnal variation in numbers of bacteria in the rumen. J. Dairy Sci. 44:1446–1456.[Abstract/Free Full Text]

Church, D. C. 1977. Livestock Feeds and Feeding. O&B Books Inc., Corvallis, OR.

Corrier, D. E., A. J. Hinton, R. L. Ziprin, R. C. Beier, and J. R. DeLoach. 1990. Effect of dietary lactose on cecal pH, bacterio-static volatile fatty acids, and Salmonella typhimurium colonization of broiler chicks. Avian Dis. 34:617–625.[CrossRef][Web of Science][Medline]

Cummings, J. H., G. T. Macfarlane, and H. N. Englyst. 2001. Prebiotic digestion and fermentation. Am. J. Clin. Nutr. 73:S415–S420.[Abstract/Free Full Text]

Donalson, L. M., W. K. Kim, C. L. Woodward, P. Herrera, L. F. Kubena, D. J. Nisbet, and S. C. Ricke. 2005. Utilizing different ratios of alfalfa and layer ration for molt induction and performance in commercial laying hens. Poult. Sci. 84:362–369.[Abstract/Free Full Text]

Donalson, L. M., J. L. McReynolds, W. K. Kim, V. I. Chalova, C. L. Woodward, L. F. Kubena, D. J. Nisbet, and S. C. Ricke. 2007. The influence of a fructooligosaccharide prebiotic combined with alfalfa molt diets on the gastrointestinal tract fermentation, Salmonella Enteritidis infection and intestinal shedding in laying hens. Poult. Sci. 87:1253–1262.

Dunkley, K. D., C. S. Dunkley, N. L. Njongmeta, T. R. Callaway, M. E. Hume, L. F. Kubena, D. J. Nisbet, and S. C. Ricke. 2007. Comparison of in vitro fermentation and molecular microbial profiles of high-fiber feed substrates incubated with chicken cecal inocula. Poult. Sci. 86:801–810.[Abstract/Free Full Text]

Durant, J. A., D. E. Corrier, J. A. Byrd, L. H. Stanker, and S. C. Ricke. 1999. Feed deprivation affects crop environment and modulates Salmonella enteritidis colonization and invasion of Leghorn hens. Appl. Environ. Microbiol. 65:1919–1923.[Abstract/Free Full Text]

Fernandez, F., M. Hinton, and B. Van Gils. 2002. Dietary man-nan-oligosaccharides and their effect on chicken caecal microflora in relation to Salmonella Enteritidis colonization. Avian Pathol. 31:49–58.[CrossRef][Web of Science][Medline]

García, J., R. Carabaño, L. Pérez-Alba, and J. C. de Blas. 2000. Effect of fiber source on cecal fermentation and nitrogen recycled through cecotrophy in rabbits. J. Anim. Sci. 78:638–646.[Abstract/Free Full Text]

Gibson, G. R., and M. B. Roberfroid. 1995. Dietary modulation of the human colonic microbiota: Introducing the concept of prebiotics. J. Nutr. 125:1401–1412.[Abstract/Free Full Text]

Guo, F. C., B. A. Williams, R. P. Kwakkel, and M. W. A. Verstegen. 2003. In vitro fermentation characteristics of two mushroom species, an herb, and their polysaccharide fractions, using chicken cecal contents as inoculum. Poult. Sci. 82:1608–1615.[Abstract/Free Full Text]

Hanson, C. H. 1972. Alfalfa Science & Technology. Am. Soc. Agron., Madison, WI.

Hohorst, J. J. 1974. Lactate. Pages 266–270 in Methods of Enzymatic Analysis. H. U. Bergmeyer, ed. Acad. Press Inc., New York, NY.

Holt, P. S. 2003. Molting and Salmonella enterica serovar Enteritidis infection: The problem and some solutions. Poult. Sci. 82:1008–1010.[Abstract/Free Full Text]

Jones, F. T., and S. C. Ricke. 2003. Symposium: Current applications, future prospects, and alternatives for the use of antimicrobials in poultry production. Poult. Sci. 82:612.[Web of Science]

Józefiak, D., A. Rutkowski, and S. A. Martin. 2004. Carbohydrate fermentation in the avian ceca: A review. Anim. Feed Sci. Technol. 113:1–15.[CrossRef]

Juskiewicz, J., Z. Zdunczyk, and J. Jankowski. 2004. Selected parameters of gastrointestinal tract metabolism of turkeys fed diets with flavomycin and different inulin content. World’s Poult. Sci. J. 60:177–185.[CrossRef][Web of Science]

Kass, M. L., P. J. Van Soest, and W. G. Pond. 1980. Utilization of dietary fiber from alfalfa by growing swine. II. Volatile fatty acid concentrations in and disappearance from the gastrointestinal tract. J. Anim. Sci. 50:192–197.[Abstract/Free Full Text]

Landers, K. L., Z. R. Howard, C. L. Woodward, S. G. Birkhold, and S. C. Ricke. 2005a. Potential of alfalfa as an alternative molt induction diet for laying hens: Egg quality and consumer acceptability. Bioresour. Technol. 96:907–911.[CrossRef][Web of Science][Medline]

Landers, K. L., R. W. Moore, C. S. Dunkley, P. Herrera, W. K. Kim, D. A. Landers, Z. R. Howard, J. L. McReynolds, J. A. Byrd, L. F. Kubena, D. J. Nisbet, and S. C. Ricke. 2007. Immunological cell and serum metabolite response of 60-week-old commercial laying hens to an alfalfa meal molt diet. Bioresour. Technol. 99:604–608.[CrossRef][Web of Science][Medline]

Landers, K. L., C. L. Woodward, X. Li, L. F. Kubena, D. J. Nisbet, and S. C. Ricke. 2005b. Alfalfa as a single dietary source for molt induction in laying hens. Bioresour. Technol. 96:565–570.[CrossRef][Web of Science][Medline]

McReynolds, J., L. Kubena, J. Byrd, R. Anderson, S. Ricke, and D. Nisbet. 2005. Evaluation of Salmonella enteritidis in molting hens after administration of an experimental chlorate product (for nine days) in the drinking water and feeding an alfalfa molt diet. Poult. Sci. 84:1186–1190.[Abstract/Free Full Text]

McReynolds, J. L., R. W. Moore, L. F. Kubena, J. A. Byrd, C. L. Woodward, D. J. Nisbet, and S. C. Ricke. 2006. Effect of various combinations of alfalfa and standard layer diet on susceptibility of laying hens to Salmonella Enteritidis during forced molt. Poult. Sci. 85:1123–1128.[Abstract/Free Full Text]

Moore, R. W., S. Y. Park, L. F. Kubena, J. A. Byrd, J. L. McReynolds, M. R. Burnham, M. E. Hume, S. G. Birkhold, D. J. Nisbet, and S. C. Ricke. 2004. Comparison of zinc acetate and propionate addition on gastrointestinal tract fermentation and susceptibility of laying hens to Salmonella enteritidis during forced molt. Poult. Sci. 83:1276–1286.[Abstract/Free Full Text]

Nisbet, D. J., D. E. Corrier, S. C. Ricke, M. E. Hume, J. A. Byrd II, and J. R. DeLoach. 1996. Cecal propionic acid as a biological indicator of the early establishment of a microbial ecosystem inhibitory to Salmonella in chicks. Anaerobe 2:345–350.[CrossRef][Web of Science]

Orban, J. I., J. A. Patterson, A. L. Sutton, and G. N. Richards. 1997. Effect of sucrose thermal oligosaccharide caramel, dietary vitamin-mineral level, and brooding temperature on growth and intestinal bacterial populations of broiler chickens. Poult. Sci. 76:482–490.[Abstract/Free Full Text]

Owens, V. N., K. A. Albrecht, and R. E. Muck. 2002. Protein degradation and fermentation characteristics of unwilted red clover and alfalfa silage harvested at various times during the day. Grass Forage Sci. 57:329–341.[CrossRef]

Patterson, J. A., and K. M. Burkholder. 2003. Application of prebiotics and probiotics in poultry production. Poult. Sci. 82:627–631.[Abstract/Free Full Text]

Ricke, S. C. 2003. The gastrointestinal tract ecology of Salmonella Enteritidis colonization in molting hens. Poult. Sci. 82:1003–1007.[Abstract/Free Full Text]

Ricke, S. C., S. Y. Park, R. W. Moore, Y. M. Kwon, C. L. Woodward, J. A. Byrd, D. J. Nisbet, and L. F. Kubena. 2004. Feeding low calcium and zinc molt diets sustains gastrointestinal fermentation and limits Salmonella enterica serovar Enteritidis colonization in laying hens. J. Food Safety 24:291–308.[CrossRef]

Rycroft, C. E., M. R. Jones, G. R. Gibson, and R. A. Rastall. 2001. A comparative in vitro evaluation of the fermentation properties of prebiotic oligosaccharides. J. Appl. Microbiol. 91:878–887.[CrossRef][Medline]

Saengkerdsub, S., W. Kim, R. C. Anderson, D. J. Nisbet, and S. C. Ricke. 2006. Effects of nitrocompounds and feedstuffs on in vitro methane production in chicken cecal contents and rumen fluid. Anaerobe 12:85–92.[CrossRef][Web of Science][Medline]

Salanitro, J. P., I. G. Blake, and P. A. Muirhead. 1974. Studies on the cecal microflora of commercial broiler chickens. Appl. Microbiol. 28:439–447.[Web of Science][Medline]

Salanitro, J. P., I. G. Blake, P. A. Muirhead, M. Maglio, and J. R. Goodman. 1978. Bacteria isolated from the duodenum, ileum, and cecum of young chicks. Appl. Environ. Microbiol. 35:782–790.[Abstract/Free Full Text]

Shermer, C. L., K. G. Maciorowski, C. A. Bailey, F. M. Byers, and S. C. Ricke. 1998. Caecal metabolites and microbial populations in chickens consuming diets containing a mined humate compound. J. Sci. Food Agric. 77:479–486.[CrossRef][Web of Science]

Sibbald, I. R. 1979. Passage of feed through the adult rooster. Poult. Sci. 58:446–459.[Web of Science][Medline]

Sibbald, I. R. 1980. Gut clearance and true metabolizable energy value of dehydrated alfalfa supplemented with sodium chloride. Poult. Sci. 59:939–940.[Web of Science][Medline]

Tsukahara, T., and K. Ushida. 2000. Effects of animal or plant protein diets on cecal fermentation in guinea pigs (Cavia porcellus), rats (Rattus norvegicus) and chicks (Gallus gallus domesticus). Comp. Biochem. Physiol. A 127:139–146.[CrossRef][Medline]

van der Wielen, P. W. J. J., S. Biesterveld, S. Notermans, H. Hofstra, B. A. P. Urlings, and F. van Knapen. 2000. Role of volatile fatty acids in development of the cecal microflora in broiler chickens during growth. Appl. Environ. Microbiol. 66:2536–2540.[Abstract/Free Full Text]

Van Soest, P. J. 1982. Nutritional Ecology of the Ruminant. O& B Books Inc., Corvallis, OR.

Woodward, C. L., Y. M. Kwon, L. F. Kubena, J. A. Byrd, R. W. Moore, D. J. Nisbet, and S. C. Ricke. 2005. Reduction of Salmonella enterica serovar Enteritidis colonization and invasion by an alfalfa diet during molt in Leghorn hens. Poult. Sci. 84:185–193.[Abstract/Free Full Text]

Xu, Z. R., C. H. Hu, M. S. Xia, X. A. Zhan, and M. Q. Wang. 2003. Effects of dietary fructooligosaccharide on digestive enzyme activities, intestinal microflora and morphology of male broilers. Poult. Sci. 82:1030–1036.[Abstract/Free Full Text]





This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Donalson, L. M.
Right arrow Articles by Ricke, S. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Donalson, L. M.
Right arrow Articles by Ricke, S. C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS