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Aloe arborescens supplementation in cat diet: evaluation of effects by in vitro gas production technique

Abstract The aim of the present study was to evaluate the effects of Aloe arborescens on organic matter digestibility (OMD), cumulative gas (OMCV) and short chain fatty acids (SCFA) production, using the in vitro gas production technique (IVGPT). Three adult cats were fed with a commercial diet (CP 31.21; EE 16.64% as fed) for 20 days before the collection of their faeces used as inoculum. The same diet, used as substrate, was incubated in vitro supplemented with different amounts (0, 0.7, 1.6 and 3.2%) of lyophilised Aloe arborescens. OMD, OMCV and SCFA significantly decreased with the increase of Aloe addition; an increase of l-lactic acid production was detected, even if pH was within physiological range. A potential prebiotic role of the Aloe arborescens carbohydrates was hypothesised in cats, but it needs further investigations. As a whole, our results show that IVGPT can represent a useful tool for nutritional evaluation of novel ingredient and/or additive also in cats.


Introduction
Since many years, the in vitro gas production technique (IVGPT) is commonly used to assess the effects of diet changes on fermentation pathways in ruminants, whereas it has been used in carnivores to study fibre source in digestive processes (Sunvold et al. 1995a). Aloe plant contains anthraquinone, glycosides, mucilages, resinous materials, sugars, mucopolysaccharides, fatty acids, glycoproteins, enzymes, vitamins and minerals (Vogler & Ernst 1999;Eshun & He 2010), thus, being particularly rich in carbohydrates, IVGPT studies can provide useful information for an in vitro screening of carbohydrates fermentability, as already shown in dogs (Cutrignelli et al. 2007;Bosch et al. 2008). Indeed, the healing properties of Aloe are due to the mucilaginous polysaccharides contained in the gel pulp, but other beneficial effects of Aloe may be due to other carbohydrates and, as a consequence to their fermentability.
As seen, most of the health benefits associated with Aloe spp. have been attributed to the polysaccharides contained in the gel of the leaf and many of its effects may derive from its activity on intestinal function, thus influencing absorption and availability of key substances (Sharma et al. 2014). However, natural products, originating from plants, have an immensely diverse array of structures, which may serve as possible lead compounds for further development into therapeutic treatments and they may be considered as an alternative to the routine pharmaceutical approach (Yarnell 2007;Eloff & Mcgaw 2008).
Proximate analysis, including total dietary fiber (TDF) evaluation (de-Oliveira et al. 2011), gives useful information on the feedstuff and diet characteristics in terms of amount of potential nutrients, but does not provide any information about their utilisation by the microorganisms of the gastrointestinal tract. Therefore, the combination of chemical composition parameters with in vitro fermentation characteristics can provide complementary information about the carbohydrates utilisation, in terms of extent and kinetics Cutrignelli et al. 2009). Therefore, the aim of this study was to evaluate the effects of Aloe arborescens supplementation on the fermentation pathway of a commercial diet for adult cat using the IVGPT.
The supplementation ratios (0.7, 1.6 and 3.2%) were calculated halving and doubling the oral dosage suggested by the producer for adult cats with 3.5 kg body weight.
The four diets were tested by in vitro gas production using cat faeces as inoculum (Sunvold et al. 1995b).
Three European adult neutered cats (3-4 years old) were used as faeces donors. All animals were healthy; a clinical visit including stool examination was performed before the onset of the experiment. The cats were progressively adapted to the standard diet [100 kcal of Metabolisable Energy/kg of Metabolic Weight (kg 0.67 )]. After 20 days, faeces were collected into thermostated boxes (39 C) under anaerobic condition. The four diets were weighed (0.5 ± 0.001 g) in 120 ml serum bottles containing a buffer solution (Bauer et al. 2001). A pool of faecal samples was diluted with NaCl solution (1:10), homogenised, filtered and added to the serum flasks (5 ml). The bottles were incubated at 39 C under anaerobic condition for 48 h. Each substrate was incubated in quadruplicate. Four bottles without substrate were used as blank.
Gas production was recorded every 2 h using a manual pressure transducer (Cole and Parmer Instrument Co, Vernon Hills, IL). After 48 h of incubation, the fermentation was stopped by cooling, and the fermenting liquor was analysed for pH (Alessandrini Instrument glass electrode; Jenway, Dunmow, UK; model 3030), short chain fatty acids (SCFA) and L-lactic acid. For SCFA determination, the sample was centrifuged twice at 12,000 g for 10 min at 4 C and 1 ml of supernatant was taken and mixed with 1 ml of oxalic acid (0.06 mol). The SCFA were measured by gas chromatography (ThermoQuest Italia SpA, Rodano, Milan, Italy; model. Focus, fused silica capillary column 30 m Â 0.25 mm Â 0.25 lm film thickness) comparing samples peaks area of each SCFA with the corresponding of an external standard composed by acetate, propionate, butyrate, iso-butyrate, valerate and iso-valerate (Calabr o et al. 2013a). L-Lactic acid was determined by spectrophotometer using a colorimetric kit (L-Lactic acid -UV method -Boehringer Mannheim/R-Biopharm. Enzymatic BioAnalysis/Food Analysis).
Organic matter digestibility (OMD) was determined by filtering under vacuum on pre-weighed glass crucibles (Scott Duran, #2) the fermentation residues, which was dried at 103 C and burned at 550 C. Gas volumes recorded during the fermentation were related to the quantity of incubated OM (organic matter cumulative volume, OMCV).
All statistical analyses were performed by SAS (SAS, 2000): the influence of Aloe addition on the in vitro parameters was tested by ANOVA, using the PROC GLM; the correlation between chemical composition data and fermentative parameters were analysed by PROC COR of the same software.

Results
The chemical composition of Aloe arborescens (CP: 15.30, EE: 1.08, Ash: 14.4, CF: 11.50% as fed) and diets (Table 1) confirm the high concentration of both structural and reserve carbohydrates. On the other hand, no differences among diets were detected for the fibre fractions with all the doses of Aloe tested.
Mean values of the in vitro organic matter disappearance, cumulative gas production and SCFA are reported in Table 2.

Discussion
Aloe arborescens showed a carbohydrate composition (TDF 29.62,IDF 21.98,SDF 7.58% as fed), which could explain the different results, obtained by IVGPT. The Aloe addition significantly (p < 0.01) reduced the OMD. Such result is probably due to the Aloe unfermentable carbohydrates content that probably affected the cumulative volume of gas produced (Sunvold et al. 1995b). This effect should be responsible for the laxative effect (Wenk 2001) of Aloe thus suggesting that the assessment of the suitable dose may be critical when using Aloe as a nutritional additive in cats. In any event, digestibility showed acceptable levels (60% OM) even after Aloe supplementation at the doses used in this trial.
The changes detected for OMD and OMCV were confirmed by a linear decrease of SCFA (Sunvold et al. 1995b), mainly due to a decrease of acetate. In contrast, a significant increase of iso-valerate, the less representative SCFA, was detected. These results may be due to changes in bacterial activity induced by Aloe supplementation. Anyway, butyrate (important energy source for the colon epithelium and regulator of cell growth and differentiation; Salminen et al. 1998) was not affected by Aloe supplementation and SCFA were always represented by more than 80% of acetate plus propionate. Both these results suggest a physiologic trend of gut fermentation .
A significant increase of L-lactic acid was detected after Aloe supplementation, thus suggesting a possible flattening of the other fermentation pathways. On the whole, these results are suggestive of a higher proliferation of lactic acid bacteria (LAB), which have been indicated as the main probiotic genera for healthy intestine of mammals (Maskell & Johnson 1993). The ability of Aloe in improving LAB activity during digestive processes may explain many of its beneficial properties (Salminen et al. 1998), but further studies focalised on the identification of gut microbiota are necessary.
The diet 3.2 showed the highest values of L-lactic acid and the lowest pH, while the lowest lactic acid concentration and the highest pH values were obtained incubating diet 0. In any event, the pH values were included in the range (5.5-7.5) indicated by Younes et al. (2001) as the physiological pH of colonic contents and resulting faeces in several animal species, including feline and canine.
The present data are in discordance with those obtained in a previous study, where the effect of Aloe supplementation was tested in vitro using rumen liquor as inoculum and diets for ruminant as substrates (Calabr o et al. 2013b). These differences are clearly due to the differences in microorganism population, thus suggesting that the IVGPT can be a useful tool to investigate gut microbiota activity also in carnivores.

Conclusions
The study of fermentation characteristics is important to understand the functional activity of innovative diets and/or feeds. The IVGPT seems particularly useful to this aim, since it allows the evaluation of both degradation extent and end-products of microorganism activities.
This study suggests that Aloe arborescens modifies fermentation pathways in cat hindgut, but the assessment of the right dosage seems to be critical to ensure a healthy effect. In conclusion, Aloe supplementation did not reveal beneficial effects in cats, but the literature in carnivores is still poor and merits further studies focalised on the identification of gut microbiota and their relation with the Aloe supplementation.