Dietary fibres may affect cats’ health, digestive processes and faecal characteristics( Reference Fahey, Flickinger, Grieshop, Van der Kamp, Asp, Miller Jones and Schaafsma 1 ). Such effects depend on the properties of dietary fibres including their potential fermentability by the intestinal microbiota. The fermentability of dietary fibres can be characterised by in vitro methods that simulate intestinal fermentation. The methodology includes incubation of the fibrous substrate of interest with a faecal inoculum from the target animal species. Various considerations for such in vitro methods have been addressed( Reference Coles, Moughan and Darragh 2 ) including the required number of faecal donors, which is described in a companion article for cats using similar laboratory procedures and substrates as in the present study( Reference Bosch, Heesen and de Melo Santos 3 ). As with any analytical method, it is important that the results obtained from an in vitro fermentation method are repeatable and reproducible. The repeatability is defined as the measure of variation for analyses within the same run, whereas reproducibility is the variation among runs( Reference Van Laar, Van Straalen and Van Gelder 4 ). Although these types of precision are routinely evaluated for standard laboratory analyses, few studies have specifically examined these for in vitro fermentation methods. This study aimed to evaluate the repeatability and reproducibility of an in vitro method for characterisation of the fermentability of dietary fibres for cats.
Experimental methods
Substrates
Dietary fibres or fibre sources were selected based on their use in cat foods, contrasting chemical composition and anticipated fermentation characteristics( Reference Flickinger and Fahey 5 – Reference Barry, Wojcicki and Bauer 7 ). Substrates were citrus pectin (CP; rapidly and highly fermentable, HM Rapid, TIC Gums), fructo-oligosaccharide (FOS; rapidly and highly fermentable; Orafti® IPS, BENEO-Orafti), guar gum (GG; rapidly and highly fermentable, 8/22, TIC Gums), molassed sugar beet pulp (SBP; slowly and highly fermentable; Research Diet Services) and wheat middlings (WM; slowly and moderately fermentable; Research Diet Services).
Animals, housing and care
A total of five neutered female European shorthair cats (3 to 5 years old), with a mean body weight of 3·4 (sd 0·4) kg were used during this 4-week study. Cats did not receive any antibiotics for at least 6 months prior to faecal collections. The cats were part of a larger colony where cats are housed in group rooms with inside and outside areas. For the detailed design of the group rooms and the climate and light schedules, see Van Rooijen et al. ( Reference Van Rooijen, Bosch and Butré 8 ). Cats were fed a nutritionally complete (i.e. meeting the FEDIAF standards) commercial dry extruded diet (Perfect Fit In-Home; Mars Petcare) for at least 4 weeks prior to the first faeces collection. Each cat was fed individually in its own metabolic cage( Reference Van Rooijen, Bosch and Butré 8 ) between 08.30 and 09.30 hours (about 45 % of their daily portion), 12.00 and 13.30 hours (about 10 %) and at 16.30 hours (about 45 %). The amount of food provided was appropriate to maintain optimal body weight. In the morning and afternoon cats went to their group room and in the evening and night cats stayed in their own cage. Water was always available ad libitum. Litter trays were only present in the metabolic cage and contained non-absorbent polyethylene litter (Katkor®; Rein Vet Products). The tray and litter were sterilised with 70 % ethanol on the day of faeces collection. The health status of the animals was monitored daily and cats were weighed weekly. The Animal Care and Use Committee of Wageningen University, Wageningen, the Netherlands Animal approved all care and experimental procedures.
Preparation of inoculum and incubation
Faeces were transferred within 15 min of defecation to sterile 250 ml plastic bottles prefilled with CO2 and 250 ml of CO2 was immediately added. The bottle with faeces was closed and transported within 5 min to the analytical laboratory where faeces were processed to inoculum under a constant stream of CO2. Faeces from cats were not pooled but processed for each cat. Attached litter particles were manually removed from faeces and faeces were diluted 1:9 (w/v) in a 39°C anaerobic sterile physiological saline solution (9 g/l NaCl). The diluted mixture was homogenised for 60 s using a hand-blender and filtered through nylon fabric (pore size 40 µm, permeability 30 %; PA 40/30, Nybolt). The filtrate was mixed with a pre-warmed (39°C) N-containing medium( Reference Williams, Bosch and Boer 9 ) in a 5:84 mixture (v/v) and gently flushed for 5 min with CO2. The resulting medium/inoculum mixture was dispensed (89 ml) into pre-warmed and CO2-flushed 250 ml serum bottles (Schott) containing 0·5 g of substrate. Bottles were then placed in the water-bath (39°C) and attached to a fully automated gas production equipment( Reference Cone, Van Gelder and Visscher 10 ), which recorded gas production for 48 h. This incubation time is longer than the average total tract transit times observed in cats (young adult cats 36 (sd 14) h; senior cats 26 (sd 6) h)( Reference Peachey, Dawson and Harper 11 ), with a orocaecal transit time of approximately 5 h( Reference Papasouliotis, Sparkes and Gruffydd-Jones 12 ). However, 48 h was estimated to be required for characterising the fermentation kinetics of SBP and WM. After 48 h of incubation, fermentation liquids were sampled for determination of SCFA (i.e. acetate, propionate, butyrate, iso-butyrate, valerate, iso-valerate) concentrations and for organic matter disappearance (only for SBP and WM). All incubations were done in triplicate. In addition, the study was repeated resulting in four runs performed over 4 weeks (one run/week).
Chemical analyses
All substrates were chemically characterised as described by Bosch et al. ( Reference Bosch, Heesen and de Melo Santos 3 ). SCFA analyses as well as organic matter disappearance were analysed as described by Bosch et al. ( Reference Bosch, Pellikaan and Rutten 13 ).
Calculations and data analyses
Gas (ml) and SCFA (mmol) productions were expressed per g organic matter (OM). Monophasic models( Reference Groot, Cone and Williams 14 ) were fitted to the data for cumulative gas production and the maximum rate of gas production (R max in ml/(g OM h)) and the time at which it occurred (T max in h) were calculated( Reference Bauer, Williams and Voigt 15 ). Acetate, propionate and butyrate were expressed as percentage of total SCFA as was the branched-chain proportion (iso-butyrate + iso-valerate). Mean and standard deviation values were calculated for all replicates of each substrate–cat combination within the four runs. CVr (standard deviation/mean × 100) were computed to describe the repeatability of the method. For the evaluation of the reproducibility, the values of replicates for each substrate–cat combination within a run were averaged and then the average for each substrate within that run was computed. The between-run mean and standard deviation values were calculated and used to compute the CVR (standard deviation/mean × 100). For production of SCFA and gas, differences among substrates within each run were tested for significance by ANOVA with Tukey pairwise comparisons.
Results and discussion
All cats remained healthy throughout the study except for one cat that produced watery diarrhoea in the week of run 2, which was not used in the study. The substrates contrasted in terms of fermentation parameters measured, as was anticipated (for parameter values, see Bosch et al. ( Reference Bosch, Heesen and de Melo Santos 3 )). Of the 285 SCFA analyses performed, six yielded false results. Fitting of the model for gas production was not possible for SBP and WM.
The total SCFA showed highly repeatable results for the more simple and pure substrate (CP, FOS, GG) whereas SBP showed a slightly higher median CVr value, and CVr value for WM was higher than 10 % (Table 1). Also the maximum CVr values were higher for the more complex substrates (SBP, WM). Acetate and propionate proportion results were highly repeatable for all substrates, but for the butyrate proportion considerably higher median CVr values were obtained. The values for branched-chain proportion were intermediate. Considerably high maximum CVr values for the proportions of SCFA were found. Outliers in SCFA profiles were not excluded in the present study, as deviating values may represent normal biological variation. For example, the proportions of acetate, propionate and butyrate for CP in the four runs were, on average, 69·1, 20·7 and 5·4 %, but in five out of fifty-six fermentation liquids the proportions were 51·1, 29·5 and 9·0 %. Such values were obtained in one of three replicates and were not associated with a specific cat or run. Furthermore, incubation with FOS, GG, SBP and WM resulted also in consistent alternative profiles. These observations may relate to selective growth of specific microbial communities with one of two distinct metabolic behaviours. This hypothesis is in line with the bimodal distribution of specific taxonomic groups found within the human faecal microbiota( Reference Lahti, Salojärvi and Salonen 16 ). For precise analysis of SCFA profile of a substrate, it is important to have at least three replicates, although additional study is required to explore these observations in more detail and determine the minimal number of replicates for precise characterisation of dietary fibre fermentability.
Coefficients of variation (%) within four runs of in vitro fermentation parameters for fibrous substrates using feline faecal inocula*
CP, citrus pectin; FOS, fructo-oligosaccharide; GG, guar gum; SBP, molassed sugar beet pulp; WM, wheat middlings; BCP, branched-chain proportion of total SCFA production; R max, maximum rate of gas production; T max, time at which R max occurred; OMD, organic matter disappearance.
* Indicated CV values (%) are based on nineteen sets of three replicates per substrate–cat combination in four incubation runs.
The CVr values for gas production and organic matter disappearance showed a similar pattern as total SCFA produced and parameters of gas production kinetics showed considerably higher values. These values were higher than those obtained by Van Laar et al. ( Reference Van Laar, Van Straalen and Van Gelder 4 ). In the latter study, CVr values were based on duplicate evaluations of four substrates (soyabean meal, wheat grain, grass silage, maize gluten meal) in three to five runs by five laboratories with rumen fluid as the inoculum source. For gas produced after 72 h, CVr values ranged between 1·4 and 10·3 % and for the parameters of gas production kinetics, similar CVr values were obtained.
The CVR values were for the measured parameters were below 10 % for the simple and pure substrates (CP, FOS, GG) and higher values were obtained for SBP and WM (Table 2). The organic matter disappearance values for SBP and WM were highly reproducible. The relatively high CVR values for R max were in line with those reported for parameters of fermentation kinetics by Van Laar et al. ( Reference Van Laar, Van Straalen and Van Gelder 4 ). The CVR values for SCFA and gas production were in line with those reported by McBurney & Thompson( Reference McBurney and Thompson 17 ) who incubated four substrates (oat bran, wheat bran, red kidney bean and GG) with faeces from one healthy human volunteer for 24 h on three separate non-defined occasions. The CVR ranged from 3 to 10 % for SCFA production whereas that for gas production ranged from 1 to 16 %. It should be noted that the reproducibility values in the present study relate to a relatively short time period applied between runs and might deviate when longer intervals are applied.
Coefficients of variation (%) between four runs of in vitro fermentation parameters for fibrous substrates using feline faecal inocula*
CP, citrus pectin; FOS, fructo-oligosaccharide; GG, guar gum; SBP, molassed sugar beet pulp; WM, wheat middlings; BCP, branched-chain proportion of total SCFA production; R max, maximum rate of gas production; T max, time at which R max occurred; OMD, organic matter disappearance.
* Indicated CV values (%) are based on four incubation runs.
Of the ten substrate comparisons within each run, consistent patterns among the four runs were found. For total SCFA, four comparisons were significant (P < 0·05) and two non-significant. For gas production this was, respectively, six and one, acetate proportion five and one, propionate proportion six and one, butyrate proportion zero and seven, and branched-chain proportion four and three. Fermentation parameters for WM were generally different from the other substrates whereas the significance of the differences between the other substrates was more inconsistent.
In conclusion, for precise dietary fibre characterisation, number of replicates should be further explored and adjusted according to the variability of the parameters of interest and the complexity of fibres. The method yielded reproducible results with some variation in obtained absolute values, which may have an impact on the significance level of the differences among substrates.
Acknowledgements
We would like to thank D. Nijkamp-Van de Pol and S. Van Laar-Schuppen for assistance during the execution of the in vitro procedures and M. Breuer for SCFA analyses.
This research was funded by Wageningen University.
G. B. contributed to all aspects of this research including research design, data collection, calculations and manuscript drafting. W. F. P., J. W. C. and W. H. H. contributed to research design, data interpretation and manuscript preparation and L. H. and K. M. S. to data collection, calculations, data interpretation and manuscript preparation.
There were no conflicts of interest.
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