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Nutrient Metabolism

Fecal Inoculum Can Be Used to Determine the Rate and Extent of In Vitro Fermentation of Dietary Fiber Sources across Three Lemur Species That Differ in Dietary Profile: Varecia variegata, Eulemur fulvus and Hapalemur griseus^{1 ,2}

J. L. Campbell^*3, C. V. Williams and J. H. Eisemann^*

^* Department of Animal Science, North Carolina State University, Raleigh NC and Duke University Primate Center, Durham, NC

³To whom correspondence should be addressed. E-mail: Jlcampbe{at}unity.ncsu.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
To estimate fermentative capacity among lemur species, four fiber substrates were tested across three species, Eulemur fulvus, Hapalemur griseus and Varecia variegata. The substrates, cellulose, beet pulp, citrus pulp and citrus pectin, ranged in composition from completely insoluble fiber (IF) to completely soluble fiber (SF), respectively. The lemurs consumed a nutritionally complete biscuit formulated for primates [85 g/100 g diet dry matter (DM)] and locally available produce (15 g/100 g diet DM). Feces were then collected and used to inoculate fermentation tubes prefilled with fiber substrates and an anaerobic growth medium. Dry matter disappearance (DMD), and acetate, propionate, butyrate, and total short-chain fatty acid (SCFA) production were measured in tubes subjected to 6, 12, 24 or 48 h of fermentation. Results were fitted to a logistic growth model. The maximal production (MP) time at which production or disappearance is at one-half maximum (t₅₀) and the fermentation rate at 3 h were calculated. The maximal disappearance of DM differed among substrates (citrus pectin > citrus pulp > beet pulp; P < 0.0001) and species (E. fulvus > H. griseus > V. variegata; P < 0.001). V. variegata reached t₅₀ for acetate and total SCFA production faster than H. griseus or E. fulvus (P < 0.02). Three-hour production rates of acetate and total SCFA were also greater for V. variegata for citrus pulp and citrus pectin (P < 0.01). Few species differences were observed for beet pulp. Results provide evidence for differences in fermentative capacity and suggest that fiber solubility and fermentability should be considered when assessing the nutritional management of lemurs.

KEY WORDS: • short-chain fatty acid production • lemurs • in vitro fermentation system • insoluble fiber • soluble fiber

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
In many organisms, the microbial flora present in either a pregastric or postgastric fermentation chamber overcome the physical barriers present in the fibrous portion of a feedstuff to break the chemical bonds between individual monosaccharide units and use them as food (1 ). Fermentability indicates the ease with which this can be accomplished and depends in part on the length of fermentation time. In vivo this is related to the retention time in the gut. Animals that rely upon short-chain fatty acids (SCFA), which are end products of this process, to contribute substantially to their total energy needs, possess adaptations to slow digesta passage through the gut and increase the time available for microbial fermentation (2 ).

Different fiber types can be characterized by their fermentability. Soluble fibers are, in general, rapidly and completely fermented due to their solubility in water and branching patterns whereas insoluble fibers such as cellulose and hemicellulose are both more slowly and less completely fermented due to their insolubility in water and, in the case of cellulose, the compact stacking patterns present (1 ). Because of differences in cell wall makeup across types of plant tissue, foods available to free-ranging herbivores will vary in the type, amount and variety of fiber present. Generally, leaves, a vegetative portion of a plant, are high in the insoluble fibers cellulose and hemicellulose and low in soluble fibers such as pectin due to their well developed cell wall. Fruits are generally lower in insoluble fiber (IF) and higher in soluble fiber (SF), making them more rapidly fermentable by gut microbes. Therefore, an animal whose diet is mainly leaf matter may possess adaptations that slow digesta passage and allow time for microbial processing of IF, whereas one that is not reliant upon IF as an energy source may simply reap some benefit from breakdown of SF, even if passage of food is rapid. Animals that consume a variable diet will likely fall somewhere in the middle, capable of subsisting on a low fiber diet but possessing sufficient modifications for some slowing of digesta passage to allow for microbial breakdown of IF when advantageous.

In an effort to improve feed efficiency, maintain animal health and expand the list of available animal feedstuffs, extensive research on the fermentability of fiber has been conducted on ruminants, pigs and horses (1 ,3 ,4 ). The fermentative capacity of cats and dogs has been examined also in an effort to determine the extent to which they can process and benefit from dietary fiber sources present in commercially available feedstuffs (3 ,5 ,6 ). The possible health benefits of fiber have also prompted research into the fermentability of the various fiber sources in human diets (7 –10 ), but little work has been conducted with nonhuman primates.

Within the lemurs, primates that possess a ceco-colic fermentation chamber, various species exhibit a variety of herbivorous feeding patterns that could result in less or more dependence upon microbial fermentation in the cecum and proximal colon. This project sought to estimate the fermentative capacity across three lemur species with differing dietary profiles, gastrointestinal tract structures and transit times (Varecia variegata, Eulemur fulvus and Hapalemur griseus) through comparisons of rate and extents of fermentation by resident microflora. The four fiber substrates tested, cellulose, beet pulp, citrus pulp and citrus pectin, range in fiber composition from predominantly IF to predominantly SF. To facilitate an across species comparison, all lemurs were offered the same diet. This minimized the confounding effect that diet itself would have on resident microflora.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Experimental design.

Four fiber substrates were tested on three lemur species using four donor animals per species. After an initial 1-wk diet transition period, all lemurs were offered the same diet for a 30-d adaptation period. After 30 d, fecal samples collected twice weekly over a 4-wk period were used to inoculate fermentation tubes filled with one of each of the four fiber substrates. Samples were to be collected from one donor animal per species per day; therefore all species would be represented for each sample collection day. Feces from each donor were collected twice to evaluate all substrates. After sample processing, fermentation tubes were subjected to a 6-, 12-, 24- or 48-h fermentation period after which time pH, dry matter disappearance and appearance of SCFA in the fermentation tubes were measured.

Substrates.

Cellulose (Solka Floc Fiber Sales and Development Corporation, St. Louis, MO), beet pulp (Michigan Sugar, Saginaw MI), citrus pulp (Freeman Industries, Tuckahoe, NY) and citrus pectin (Sigma-Aldrich, St. Louis, MO) were the fiber substrates used in this experiment.

Substrate analysis.

Substrate samples were analyzed in duplicate. Dry matter was determined by placement in a forced air oven at 105°C for 24 h. Samples were ashed in a muffle furnace at 550°C for 4 h, and organic matter was determined by subtracting ash value from total dry matter (11 ). The IF, SF and total dietary fiber (TDF) were determined by the methods outlined by Prosky et al. (12 ,13 ). Nitrogen was determined by Kjeldal analysis (11 ). Table 1 shows the chemical compositions of the fiber substrates.

The Duke University Institutional Animal Care and Use Committee approved use of animals for this project. Four lemurs from each of the following three species, H. griseus, V. variegata and E. fulvus were selected from the colony at Duke University Primate Center (Durham, NC) to use as fecal donors. Selection was based on animal availability; however, lemurs of similar ages were used when possible (Table 2 ). They were housed as male-female pairs in outdoor cages ranging from 2.7 x 1.9 x 3.4 (length x width x height) m to 3.5 x 4.1 x 6.9 m in size.

Sample collection and processing.

Animal donors designated for collection on a given day were observed for 1 h beginning at sunrise. If no sample was produced within that time period, another animal of the same species was sampled if possible to expedite processing of samples collected from other donors. The animal missed was then sampled at a later date. All sample days included feces collected from at least two species so those samples from one species were not the only ones collected on sample day. The fresh sample was immediately sealed in a waterproof plastic bag and placed in a prewarmed (37°C) thermos for transport to the laboratory.

Upon arrival in the laboratory, samples were diluted 1:10 in an anaerobic dilution solution prewarmed to 37°C and blended for 15 s in a blender (14 ). This solution was then filtered through four layers of cheesecloth, and the filtrate was sealed in 125-mL bottles under CO₂. Warmed plastic 50-mL centrifuge tubes, prefilled with 310 mg of fiber substrate and 30 mL of an anaerobic growth medium (3 ), were inoculated with 1 mL of the diluted feces. Blank tubes consisted of medium and 1 mL of inoculant, but no substrate was added. Tubes were immediately flushed with anaerobic grade CO₂, capped with one-way release valve stoppers, agitated gently and placed in an incubator (Fisher Isotemp Incubator; Fisher Scientific, Pittsburgh, PA) set at 37°C. Fecal samples from each animal and blanks were run in triplicate for each of four time periods, 6-, 12-, 24- and 48-h incubation. Based on previous publications (3 ), the assumption was made that for cellulose, bacteria would not have had enough time in 6 h to achieve adequate numbers to metabolize cellulose into SCFA; therefore tubes for the 6-h time period were not prepared. At the end of each time period, designated tubes were removed from the incubator and pH was taken with a pH meter (Acumet Portable Meter; Fisher Scientific). Aliquots (4 mL) of the unstirred layer were then taken and prepared for SCFA analysis.

The remaining solution, used to determine dry matter disappearance, was combined with 100 mL 95% ethanol and allowed to precipitate at room temperature for 1 h. Samples were then filtered through ash-free Whatman filter paper (Whatman International, Maidstone, UK) under a mild vacuum and sequentially rinsed with three 10-mL portions each of 78% ethanol and 95% ethanol, and two 10-mL portions of acetone. Samples were placed under a hood to air dry and then dried in an oven at 105°C for 6 h. DMD was determined as 1-(DM sample residue - DM blank residue)/original DM) x 100. Samples were not corrected for the 4-mL aliquot removed.

The 4-mL aliquot intended for SCFA analysis was placed in a labeled centrifuge tube, mixed immediately with 1 mL of 25% meta-Phosphoric Acid, and allowed to precipitate for 20 min at room temperature. Tubes were then centrifuged at 13,500 x g at 6°C for 20 min. The supernatant fraction was collected, placed in a labeled storage tube and frozen at -20°C until analysis. After thawing, samples were centrifuged at 9000 x g at 6°C for 5 min, and the supernatant was collected for SCFA analysis. The sample (1 mL) was placed in a gas chromatography vial along with 100 µL of an internal standard (2-ethyl butyrate, 1.16 mmol/L), and SCFA concentration in the sample was determined with a Varian 3800 Gas Chromatograph (Varian Chromatography Systems, Walnut Creek, CA). A Nukol capillary column (Supelco, Bellefonte, PA) was used, and nitrogen was the carrier gas at 0.508 kPa. The injection port and detector port temperatures were both 250°C. The initial oven temperature was 80°C, programmed to rise 20°C/min to 140°C, held at 140°C for 1.75 min, then programmed to rise 30°C/min to 175°C and held for 10 s. Total run time was 6 min, and the split ratio at injection was 1:25.

Data analysis.

Data at different fermentation times were analyzed as a repeated measures design using the MIXED model procedure of SAS (15 ). Species, incubation time, substrate and all interactions were tested, and day and day x species x substrate were used as random effects in the model. Incubation time was used in the repeated statement. Least significant difference was used to determine differences between individual means.

Acetate, propionate and total SCFA production and dry matter disappearance data were fitted to a logistic growth model (16 ) using the nonlinear mixed procedure of SAS (SAS Statistical Software, Cary, NC).

The logistic model used was

where F = the quantity of SCFA produced or DM disappearance at time t, MP = maximal production or disappearance, k = fractional rate constant, t = in vitro fermentation time and t₅₀ = time at which SCFA production or DM disappearance is half of maximum. Estimates of MP, k and t₅₀ were made for each donor. Production or disappearance rates at 3 h were estimated by solving the equation above for t = 3 h and substituting the value obtained for F into the following rate equation:

Analysis of the fermentation rate at 3 h was chosen with respect to reported transit time data for two of the species tested. The model-generated estimates for MP, t₅₀ and production or disappearance rate at 3 h were analyzed as a completely randomized design using the MIXED model procedure of SAS (15 ). Least significant difference was used to determine differences between individual means. For all tests, the level of significant difference was P < 0.05. Values in text are means ± SEM.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Lemurs were housed as pairs, thus dietary intake data were calculated for two lemurs. All pairs remained healthy, maintained constant daily dietary intakes and produced normal feces throughout the experiment. One female H. griseus within a pair lost weight over the 45-d period. The V. variegata, E. fulvus and H. griseus pairs consumed 50.39 ± 0.61 g/kg body weight^0.75, 45.65 ± 1.79 g/kg body weight^0.75 and 33.94 ± 2.36 g/kg body weight^0.75 of DM daily, respectively.

The pH of medium following fermentation did not differ among species; however, there was a substrate x time interaction (Table 3 ; P < 0.0001). For citrus pectin, citrus pulp and beet pulp, the pH dropped most between the 6- and 12-h times. At the 24- and 48-h time periods, pH values were lowest for citrus pectin, intermediate for beet pulp and citrus pulp and highest for cellulose.

View larger version (27K): [in this window] [in a new window]   FIGURE 1 Dry matter disappearance and SCFA production versus in vitro fermentation time for H. griseus (), V. variegata () and E. fulvus () for three fiber substrates, beet pulp, citrus pulp and citrus pectin. Data points represent species means. The pooled SEM for dry matter disappearance (DMD), acetate production, propionate production and total short-chain fatty acid (SCFA) production were 4.0 mg · g-1 of original substrate, 0.17 mmol acetate · g-1 of original substrate, 0.06 mmol propionate · g-1 of original substrate, and 0.22 mmol total SCFA · g-1 of original substrate, respectively. Curves represent fit to a logistic growth model (16 ).

Main effect means of substrate and species for propionate maximal production and t₅₀ are shown in Table 4 . Maximal production of propionate also showed a substrate x species interaction (P < 0.05). H. griseus produced less propionate in the beet pulp-filled tubes (1.32 ± 0.05 mmol · g of original substrate^-1) than the other two species, which did not differ (1.69 ± 0.05 mmol · g of original substrate^-1). Propionate productions from citrus pulp were similar, with H. griseus producing less (1.02 ± 0.05 mmol · g of original substrate^-1) than V. variegata and E. fulvus (1.31 ± 0.05 and 1.36 ± 0.05 mmol · g of original substrate^-1, respectively). As for acetate, the t₅₀ of citrus pulp was earliest among substrates whereas beet pulp reached t₅₀ last (P < 0.0001). No species differences were observed for the t₅₀ of propionate. Production rates at 3 h showed significant substrate x species interactions (P < 0.001; Table 5 ). For beet pulp and citrus pulp, H. griseus had the lowest rate at 3 h and the other two species had more rapid rates, which did not differ. For pectin, V. variegata showed the highest rate and the other two species had slower rates, which did not differ.

Maximal production of total SCFA also differed among substrates (Table 4) . Beet pulp produced the greatest amount of SCFA, and citrus pulp produced the least P < 0.0001). Substrate and species both affected t₅₀. Among substrates, calculated t₅₀ patterns were the same as for other SCFA, citrus pulp achieved t₅₀ most rapidly at 10 h post-inoculation whereas beet pulp was the slowest at 17 h (P < 0.0001). Among the species, V. variegata reached t₅₀ 2 h earlier than H. griseus and E. fulvus (P < 0.02). Production rates at 3-h post-inoculation had a substrate x species interaction (Table 5) . V. variegata production rates were higher than the other two species for citrus pulp and citrus pectin (P < 0.01). Beet pulp showed a similar trend (P < 0.07).

Overall, butyrate production was low so the data were not included in curve fitting. However data obtained from 6, 12, 24 and 48 h indicated that butyrate appearance had species x substrate (P < 0.001) and substrate x time (P < 0.0001) interactions. At 48 h, fecal inoculum from H. griseus (0.203 ± 0.02 mmol · g of original substrate^-1) produced less butyrate than V. variegata (0.275 ± 0.02 mmol · g of original substrate^-1) and E. fulvus (0.280 ± 0.02 mmol · g of original substrate^-1) when the substrate was citrus pulp. For citrus pectin, V. variegata (0.297 ± 0.02 mmol · g of original substrate^-1) produced more butyrate than E. fulvus (0.164 ± 0.02 mmol · g of original substrate^-1) and H. griseus (0.207 ± 0.02 mmol · g of original substrate^-1). Butyrate levels were lower for beet pulp at 12 h than for citrus pulp, but similar to citrus pectin. At 48 h, butyrate levels did not differ among all substrates; however, a plateau had not been reached for citrus pulp or beet pulp.

The acetate/propionate ratios obtained from maximal production estimates differed among species (P < 0.0002) and substrates (P < 0.0001). Among substrates, citrus pectin had the highest ratio (6.24 ± 0.10 to 1), and beet pulp the lowest (2.76 ± 0.10 to 1). Among species, H. griseus had the highest ratio (4.69 ± 0.11 to 1) whereas V. variegata and E. fulvus were lower and did not differ (3.59 ± 0.11 to 1 and 3.91 ± 0.11 to 1).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Most mammals possess the microflora necessary for the fermentation of fiber, and many have developed specializations that enhance the recovery of SCFA from microbial fermentation. Because microorganisms require certain living conditions as well as time to process the fiber present, the host provides specific environmental conditions (substrate, temperature, pH) as well as the time necessary to process the fiber. Ambient conditions will favor one population over another. Most fiberolytic bacteria that consume IF substrates such as cellulose and hemicellulose prefer a pH range of 6.0–6.5 at a temperature of 37°C. Turnover rates of bacterial populations are highly dependent on environment, and changes in the type of fiber in the diet can quickly alter the microbial population. Regardless, anatomical adaptations such as a large capacious cecum and colon suggest reliance upon those fiberolytic microbes that prefer IF substrates, since this is true for species that possess these adaptations and for which the microbial population is known (1 ,2 ). These types of adaptations exist to varying degrees among diurnal lemur species, and based on these results lemurs possess the microbial flora necessary to ferment large amounts of the fibrous portion of their diet.

Across many species, the absorbed SCFA have different fates. Butyrate is generally metabolized by the enterocytes themselves, whereas the liver takes up propionate for use in carbohydrate metabolism as necessary. Acetate bypasses both the gut and liver and is used for fuel by peripheral tissues (1 ). Thus differences in the fiber makeup of diets consumed by wild lemurs will govern the relative amounts of SCFA produced and availability for these varied metabolic pathways. For example, the pectins in fruit produce a higher proportion of acetate relative to propionate; therefore, the majority of that energy will not be used by the liver to produce glucose but rather by the peripheral tissues for fuel whereas the more slowly fermented cellulose will produce relatively more propionate for glucose production.

The cellulose used in this experiment was a highly purified and crystalline form. The high percentage of TDF, all of which was IF, made this substrate by far the least fermentable of the sources used in this study and this was reflected in the very low disappearances recorded at all time periods by all species. These results were similar to those reported by Sunvold et al. (1 ), where little fermentation of purified cellulose occurred across a variety of species. The citrus pectin used was also a purified form, rather than a commercially available source that contained large amounts of soluble sugars. Therefore, early actual values for DMD and SCFA production were lower than observed in other work where commercial sources were used; however, values at later time periods were similar (3 ,9 ). Substrate samples were well hydrated with the anaerobic medium in advance of inoculation; therefore, the lag time was likely due to the time required for microbes to increase in number and penetrate the gel matrix formed by the citrus pectin. Once accomplished, fermentation of the SF present was rapid and complete. Bourquin et al. (9 ) determined that the water holding capacity of unfermented citrus pectin was high, but once fermented it did not differ from a variety of other fermented fiber substrates, an indication that the SF present had been fermented completely.

Citrus pectin yielded the highest ratio of acetate to propionate. Pectin is composed primarily of galacturonic acid units of which acetate is the most common degradation product (1 ) so this result was expected. Pectins serve as cementing and hydrating agents and are present in highest concentrations in plant tissues that are growing rapidly and high in moisture content, thus fruits can be a good source of pectins.

Citrus pulp, a by-product of orange juice extraction, is composed of pectin and cellulose. The citrus pulp used in this study contained 436 mg · g SF^-1, or pectin and 418 mg · g IF^-1. The beet pulp used in this study had 130 mg · g SF^-1 and 689 mg · g IF^-1. Beet pulp is the residue from sugar extraction and is considered to be a relatively low cost animal feed (17 ). The pectin isolated from beet pulp is lower in molecular weight and lower in gelling capability than that found in citrus or apple pectin sources (18 ). The carbohydrate portion of beet pulp has been fractionated into 250 mg · g galacturonic acids^-1 from pectin, 320 mg · g hemicellulose^-1 (xylose, mannose, glucose, galactose and arabinose), 300 mg · g cellulose^-1, and the remainder neutral sugars (19 ). Both the beet pulp and the citrus pulp are mixed fiber substrates and therefore better represent an animal’s true fermentative capacity. It is interesting that the beet pulp, containing a greater portion of IF, was processed similarly across species, whereas species differences were observed for the citrus pulp, a substrate relatively higher in SF. This may suggest that differences in fermentative capacity among these species are more evident for the rapidly fermented pectins and gums, possibly because of the bacteria species and numbers present. However this could also be a limitation of the in vitro system, in that anticipated differences in IF disappearance over time were not evident because of the artificial environment in the fermentation tubes.

Although not always true for some individual SCFA values, the MP of total SCFA was similar among the lemur species. The three species combined showed similar responses to time and substrate as have been observed for other species. Values from humans (10 ) and from pigs, horses, cattle, dogs and cats (3 ,5 ,6 ) for DMD and acetate, propionate, butyrate and total SCFA production were all comparable. Previous researchers suggested that perhaps the similarities among species in capacity for extent of microbial fermentation in an in vitro system have less importance in vivo than the rate of fermentation at the earlier time periods, in that animals with shorter transit times would have a lower extent of fermentation than those animals with longer transit times. If true then differences observed in this project, when discussed in terms of what is known about transit times across species, imply that the different species would vary in their capacity to obtain SCFA from fiber sources. For example, results from these data suggest V. variegata could indeed use the SF present in foods as a source of SCFA; however, production will be highly dependent on rate of fermentation. Published transit times for V. variegata are 2.3 to 5.2 h for a variety of marker types (20 ,21 ), providing V. variegata with only a short time for microbial processing. The t₅₀ values obtained for V. variegata were shorter than for the other two species for acetate and total SCFA and tended to be shorter for propionate and DMD. More importantly, V. variegata also showed faster rates of acetate and total SCFA production at 3 h for citrus pulp and citrus pectin, suggesting a capacity to more rapidly process the fiber present in these substrates. Generally, maximal production of SCFA for V. variegata did not differ from H. griseus and E. fulvus, and maximal disappearance of DM was less, so differences observed for V. variegata, which suggest enhanced fermentation, were related to early rates of fermentation and not extent of production. The diet of free ranging V. variegata consists of large amounts of ripe fruit with low to moderate amounts of leaf consumption seasonally (22 ). The rapidly degradable SF portions of the fruit can therefore be a useful source of energy for these animals, although their contribution will be highly dependent on fermentation rate due to minimal retention time.

Recorded transit times for E. fulvus range from 2.7 to 4.6 h (23 ,24 ), similar to values obtained for V. variegata. Estimated production rates of total SCFA at 3 h for this species were similar to the other two species for beet pulp, lower than V. variegata but similar to H. griseus for citrus pectin, and lower than V. variegata but still almost twice the rate of H. griseus for citrus pulp. Feeding ecology studies indicate that E. fulvus consumes food items across a wide range of fiber quantity and type (25 –27 ). Thus, perhaps E. fulvus does not have the enhanced capacity to process SF rapidly as V. variegata, but it could reap some benefit. Markers used to determine transit times for E. fulvus thus far are not ideal estimates of digesta passage; therefore, more in vivo data would clarify these results. It is likely that fermentation rate would determine the degree to which E. fulvus could use SCFA as a source of fuel, and these results suggest a low yield of SCFA and a greater dependence upon nonfibrous carbohydrates.

Sample tubes inoculated with H. griseus showed similar or lower MP values than other species and longer t₅₀ times than V. variegata for some variables. Production rates of total SCFA were lower than V. variegata and E. fulvus for citrus pulp, similar to E. fulvus for citrus pectin but similar to both species for beet pulp, the mixed substrate with the greater proportion of IF. Because transit times recorded for H. griseus range from 18.2 to 39.3 h, depending on marker type (24 ,28 ), maximal production of SCFA, and not rate at early time points, has more relevance. These long transit times would provide H. griseus with more opportunity to process the fibrous portion of a foodstuff; therefore, H. griseus could gain more from consumption of an IF source compared with V. variegata, especially if hemicellulose were present in high quantities compared with cellulose. The bamboo diet of H. griseus (29 ,30 ) is high in IF, which would require a longer residence time for processing relative to fruits, and is high also in hemicellulose. Bamboo, a monocot, is low in the SF pectin (18 ), thus more rapid fermentation of SF may not be necessary in animals that consume bamboo diet. For this species then, the extent of fermentation and not rate may have a greater impact on the degree to which microbial fermentation contributes such that a substrate with a low rate of fermentation could still make a substantial contribution to the energy budget.

These data provide a good preliminary discussion of the idea that differences in anatomy and physiology, in response to different dietary profiles, may result in differences in the capacity to process different forms of dietary fiber. Data from both V. variegata and H. griseus fit well with this concept, whereas data from E. fulvus are less clear. Lemurs were fed a similar diet to eliminate diet as a factor and focus instead on possible differences related to anatomy and motility within the gut. In vivo research, such as direct comparison of digestibility and digesta passage, will improve the degree to which these results can be interpreted. Further in vitro studies using mixed diets are also warranted. Nonetheless, all three species are capable of microbial fermentation and their in vitro fermentative capacity appears to coincide well with dietary profiles for at least two of the species. V. variegata can benefit from the rapid fermentation of SF whereas H. griseus increases fermentation by prolonging residence time in the gut. E. fulvus may be more flexible, depending on the type of fiber present in the diet, but more data on in vivo transit time and fermentation using a variety of mixed substrates are required before inferences can be made.

An in vitro fermentation system was used in this project because it is a low cost and low risk method of estimating fermentative capacity for a variety of species and substrate types. It is also ideal for a zoo setting, in that animals can remain in normal housing, sample collection is simple and stress to the animal is minimal. These data provide information useful for assessment of species differences. However, these results indicate that the data should, when possible, be considered with respect to in vivo measurements such as digestibility and transit time to determine the true biological value of SCFA production by resident microflora. These data suggest that there are differences among lemur species; therefore, fiber type should be considered along with quantity when designing appropriate captive diets. For example, captive V. variegata and E. fulvus may benefit from a diet containing fiber sources that provide an optimal balance of both IF and SF components whereas a diet whose main source of fiber is composed of IF may be more appropriate for H. griseus. What constitutes an optimal balance of fiber types for E. fulvus and V. variegata, or the ideal quantity of fiber in diets for all three species is unclear; however, further use of both in vitro systems to test other mixed fiber substrates and in vivo testing of diets containing differing fiber sources could answer these questions.

	ACKNOWLEDGMENTS

 
We thank the staff of Duke University Primate Center for their support of this project, and Robin Pelletier, Carol Holman, Joni Meredith, and Thomas Ross for their help in data collection and laboratory analyses.

	FOOTNOTES

 
¹ Presented in part at the Annual Meeting of the AZA Nutrition Advisory Group, September, 2001, Orlando FL [Campbell, J. L., Eisemann, J. H. & Williams, C. V. (2001) The use of fecal inoculum to determine the rate and extent of in vitro fermentation for cellulose, beet pulp, citrus pulp and citrus pectin across three lemur species: Varecia variegata, Eulemur fulvus, and Hapalemur griseus. In: Proceedings of the American Zoo and Aquarium Association Nutrition Advisory Group 4th Conference on Zoo and Wildlife Nutrition (Edwards, M. S., Lisi, K. J., Schelgel, M. L. & Bray, R. E., eds.), pp. 29–36, AZA Nutrition Advisory Group.].

² Project funding was provided in part by the North Carolina Agricultural Research Service. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service or criticism of similar products not mentioned.

⁴ Abbreviations used: DM, dry matter; DMD, dry matter disappearance; IF, insoluble fiber, MP, maximal production, SCFA, short-chain fatty acids; SF, soluble fiber; TDF, total dietary fiber; t₅₀, time at which production or disappearance is at one-half of maximum.

Manuscript received 1 March 2002. Initial review completed 3 April 2002. Revision accepted 28 June 2002.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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3. Sunvold, G. D., Hussein, H. S., Fahey, G. C., Merchen, N. R. & Reinhart, G. A. (1995) In vitro fermentation of cellulose, beet pulp, citrus pulp, and citrus pectin using fecal inoculum from cats, dogs, horses, humans, and pigs and ruminal fluid from cattle. J. Anim. Sci. 73:3639-3648.[Abstract]

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