EFFECT OF NON STARCH POLYSACCHARIDES OF CEREAL GRAINS ON INTESTINAL MICROFLORA OF CHICKENS AND DUCKS

Andrzej Wiliczkiewicz
Department of Animal Nutrition and Feed Quality, Wrocław University of Environmental and Life Sciences, Poland

ABSTRACT

The influence of the type of non-starch polysaccharides on the microflora composition of the contents of small intestine and caecum of poultry was investigated in the conducted experiment. Four cereal grain types (maize, wheat, barley and oat), being a basic source of polysaccharide differentiation in the diets of chickens and geese, constituted the subject of the study. A total number of aerobic bacteria, lactic acid bacteria as well as yeasts and moulds were determined in the contents of ileum and caecum of the birds. Besides, an interdependence between the contents of structural carbohydrates, SCFA concentration, pH value and microflora composition of the analysed alimentary tract segments was investigated.

The using of ground wheat instead of maize as the main diet components resulted in the increase of xylose contents in them, and in case of ground barley and oat it also caused the increase of crude fibre, NDF, hemicelluloses and cellulose. These change caused the increase of the general number of aerobic bacteria and lactic acid bacteria in the contents of ileum and caecum of the birds. A higher number of bacteria was found in ducks in ileum contents in the groups receiving concentrates with the increased share of structural carbohydrates in comparison with chickens, where a similar tendency was not observed. A higher general number of aerobic bacteria and lactic acid bacteria were found in chickens when compared with ducks. The contents of structural carbohydrates, microflora composition, SCFA concentration as well as pH reaction in the contents of ileum and caecum were mutually correlated (p≤0.01).

Key words: non-starch polysaccharides, cereal grains, chickens, ducks, intestinal microflora.

INTRODUCTION

Cereal grains constitute basic components of fodder concentrates for poultry. They include significant amounts of non-starch polysaccharides, in the range from 9.7 in case of maize, up to 23.2% of the dry matter in oat grain [4]. These carbohydrates are not digested by endogenous enzymes of monogastric animals; they can, however, be degraded by microorganisms in the lower segments of the alimentary tract of birds. Undigested nutrients in the intestinal contents constitute the main source of energy and components necessary for the growth of multiplication of the intestinal flora. The alimentary tract microorganisms compete against the host organism using alimentary components, which could be used by the host [6, 12]. Particular bacteria species prefer various substrates serving for their development. The composition of the diet as well as the concentration of alimentary components in it influence the intestinal flora and, in this way, affect the animals’ ability to digest and absorb the nutrition components [19]. The chemical composition and structure of the alimentary tract contents determine the proportions between bacteria species [22].

The bacterial flora have a important role in the digestion physiology. The enzymes produced by bacteria facilitate the digestion of alimentary components, including carbohydrates [13, 18].

MATERIALS AND METHODS

The experiments commenced on 21-day old broiler chickens (Vedette hybrids) and muscovy ducks (Grimaud hybrids), which were randomly allocated to 4 experimental groups. The relation of sex in both species was approximately 1 : 1. The birds, 75 animals of each species, were kept in balance cages in groups of five animals. The animals of each groups were fed with complete concentrates with a dominant contribution of ground maize (48%) – group I – control group, ground wheat (65%) – group II, ground barley (45%) – group III and ground oat (20%) – group IV (table 1). The following system of replication was kept for each species: 3 replication in the control group and 4 replications in the remaining experimental groups, a total of 30 replication. The mixture recipes were formed in a way enabling the crude fibre contents originating from the investigated components to be approximately 20 g×kg^-1 of the mixture. The energy value of the applied feeding mixtures was similar and on average amounted to 11.6 MJ×kg^-1, while the protein contents was close to 19%.

On the last day of the eighth and tenth week of the birds’ life, two hours after feeding, 3 or 2 animals were selected for dissection from each cage. Directly after slaughter, samples were collected from the terminal part (about 25 cm) of ileum and both caeca including about 2 g of the intestinal contents. The samples in sterile packaging were transferred to microbiological assays. The remaining segments of the intestines were turned inside out in order to collect their contents. The pH reaction of the obtained intestine contents was determined, and then samples were taken for the purpose of determining the short-chain fatty acids (SCFA) and dry matter. Crude fibre, neutral detergent fibre (NDF), and acid detergent fibre (ADF) were determined in the dried samples of the digesta.

The chemical components of dietary components in fodder and intestinal contents were assayed by means of standard methods. In case of the dry matter, total protein and crude fibre (Henneberg-Stohman method – with the application of Fibertec apparatus manufactured by Tecator), the methods consistent with AOAC were used [1]. The determination of NDF, ADF and acid detergent lignin (ADL) were conducted with Van Soest method [26]. The hemicellulose value was calculated as a difference between NDF and ADF, while cellulose – between ADF and ADL. The total dietary fibre (TDF) contents with a distribution into insoluble (IDF) and soluble (SDF) fraction was determined according to Asp et al. method [3].

The pH measurements of intestinal contents were conducted with potentiometric method with the application of Medical Mat 1202 SM pH-meter. In order to determine SCFA, 0.5 ml of concentrated orthophosphoric acid, 0.5 ml of acetic acid and 2 ml of water were added to 2 g of the intestinal contents. Then, the sample was mixed and centrifuged. The separation of fatty acids was carried out in supernatant with the use of PYE UNICAM-104 gas chromatograph with 27-cm column.

The microorganisms in investigated segments of the birds’ alimentary tract were determined by weighing 1 g of the intestinal contents into 9 ml of buffered peptone water. The sample prepared in this way was homogenised in Lab-Blender 400 stomacher for two minutes. Then, subsequent decimal dilutions were prepared. Cultures of 0.1 ml of suspension were put on solid bacteriological media out of particular dilutions. The general number of bacteria was determined by means of the NPL method (method of the most likely number) on TSB medium. The examined material was incubated at the temperature of 37°C, and the reading was taken after 72 hours. The lactic acid bacteria were determined on MRS medium, incubated at the temperature of 30°C, and the reading was taken after 72 hours. Yeasts and moulds were determined with the use of yeast-peptone-glucose agar with chloramphenicol (Chloramphenicol Yeast Glucose Agar) addition as a medium. The cultured material with the medium was incubated at the temperature of approximately 25°C, and the result was read after 7 days.

The experiment results were analysed with statistical methods with the use of a two factor variance analysis according to the following model:

y_ijk = μ + α_i + β_j + (αβ)_ij + ε_ijk

where y_ijk – denotes the value of the observed dependent variable, μ – general variable, α_i – influence of diet, β_j – influence of bird species, (αβ)_ij – interaction between type of diet and bird species, ε_ijk – residue error. The numerical material was transformed by means of a logarithmic method (log₁₀) prior to further analysis. The significance of differences between the mean values was determined with Tuckey’s test. Pearson’s correlation coefficients were calculated for the basic parameters determined in the intestinal contents, independently of their segment examined, while their significance was assessed with t test. The calculations were conducted with the use of Statistica 6.0 [23] software. The test results regarding the microflora composition of the alimentary tract for the main factors (source of NSP and bird species) were presented in tables, while intermediate values (within both factors) in a graphic form.

RESULTS AND DISCUSSION

Using of various cereal grain components in experimental mixtures resulted in the differentiation of the carbohydrates fraction (table 2). The concentration of crude fibre in the birds’ mixtures ranged from 3.2% in group II (ground wheat) up to 4.6% in group IV, whose basic component was constituted by ground oat. The NDF level in the tested mixtures was similar and amounted to, on average, 12.4% in the mixtures with ground maize and wheat, while in the mixtures with the addition of ground barley and oat it was 14.2%. The ADF contents in the control mixture (I) amounted to 4.3%, while in fodder including barley and oat its level was higher by 18 and 44%. The mixture of group III was characterised by higher, approximately by 12%, contents of hemicelluloses, in comparison with the mixtures administered to the remaining groups of animals. The mixture of group IV (ground oat) included the highest amount of cellulose – 3.7%.

Moreover, one should pay attention to xylose contents, higher – on average – by 23% in mixtures of groups II, III and IV in comparison with the control group (I), where its contents amounted to 2.8%. The birds assigned to group III received a fodder mixture including the highest contents of b-glucans (1.8%) and dietary fibre (15.9%), while the animals of group I and II had about 13% of this component at their disposal in the mixtures. The participation of 20% of ground oat into the mixture (IV) increased the amount of dietary fibre by approximately 16% in comparison with the control group.

The mean body weight of 21-day old birds assigned to the experimental groups amounted to 0.45 kg, 2.5 kg in the eighth week and 3.3 kg in the tenth week. The initial mean body weight of chickens was 0.63 kg, ducks 0.27 kg, while at end of the last balance – 3.70 and 2.92 kg, respectively. The fodder intake in the period directly preceding the birds’ slaughter was similar in the all tested dietary groups, and in the eighth week of life it was on average 134, while in the tenth week – 225 g/day/animal.

According to various authors, the general number of aerobic bacteria in the contents of ileum can be from 5.5 up to 7.3 log cfu×g^-1 [5, 20, 25]. Hűbener et al. [7] observed the increase of the number of intestinal microorganisms in poultry fed with fodder of high non-starch polysaccharide contents. A similar effect was obtained through the addition of cereal grains with higher structural carbohydrates contents into the birds’ diet in the presented experiment. The lowest general number of aerobic bacteria in ileum contents was observed (table 3) in the group of birds fed the control mixture (I) (7.25 log cfu×g^-1 g). These values were higher in the remaining experimental groups, and amounted from 8.25 (II) up to 8.50 log cfu×g^-1 (IV). The increase resulted from higher values obtained in ducks, while the mean values of this parameter in experimental groups of chickens were similar to the control group (fig. 1). The highest number of aerobic bacteria occurs in caecum, and the values determined in chickens in this intestine range from 7.7 up to 11.8 log cfu×g^-1 [5, 8, 20]. The measurements conducted within the scope of this study were contained within these limits. The general number of bacteria in caecum was even in group I, II and IV (from 9.13 up to 9.25), while in group III, which was administered a mixture with a higher amount of ground barley, the mean value amounted to 9.71 log cfu×g^-1. Higher number of these bacteria in the intestinal contents of chickens was determined in the groups receiving mixtures of a higher concentration of structural carbohydrates, while no similar tendency was observed in ducks (fig. 2). The general number of bacteria determined in the intestinal contents was lower in chickens when compared with ducks, and the mean values in ileum in both bird species were 7.50 and 8.69 log cfu×g^-1, and in caecum – 9.20 and 9.44 log cfu×g^-1, respectively.

The highest number of lactic acid bacteria occurs in chickens’ caecum, and the values determined by other authors were contained in the range from 5.8 to 9.9 log cfu×g^-1 [14, 25], while in the small intestine – from 4.2 up to 8.8 cfu×g^-1 [5, 8, 21]. The number of lactic acid bacteria determined in this experiment in the contents of ileum of the birds of groups I and IV was 7.38 cfu×g^-1, and in groups II and III – 7.75 and 7.88 log cfu×g^-1, respectively. The highest number of lactic acid bacteria was found in caecum of the birds of group IV – 8.75 log cfu×g^-1, and in the remaining cases – from 8.00 up to 8.29 log cfu×g^-1. A similar effect was obtained by Choct [unpublished data, after 2] who administered barley to chickens in their diets and observed an increase (p≤0.05) of Lactobacillus bacteria in caecum, while the presence of oat in the diets favoured the development of Escherichia and Lactococcus bacteria. Also Hűbener et al. [7], Jamroz et al. [10, 11] as well as Mathlouthi et al. [16] observed the increase of lactic acid bacteria number in the contents of caecum and ileum by substituting maize with wheat and barley in concentrates for birds. This type of bacteria was present in a higher quantity in ileum of ducks (8.00 log cfu×g^-1) when compared with chickens (7.50 log cfu×g^-1), where the differences between the mean values of dietary groups were insignificant (fig. 3). The number of lactic acid bacteria in the contents of caecum was similar in both bird species and on average amounted to 8.29 log cfu×g^-1.

The number of yeasts and moulds in ileum contents differed to a higher degree between chickens and ducks – 2.77 and 2.15 log cfu×g^-1, however the mean values in the feeding groups were similar and amounted from 2.25 log cfu×g^-1 (III) up to 2.55 log cfu×g^-1 (IV). The number of fungi in caecum contents of 21-day old chickens fed with a diet including ground maize was 3.0 log cfu×g^-1, and when the ground maize was substituted with ground wheat and barley, the number increased up to 4.4 log cfu×g^-1 [9]. On the other hand, the same procedure in older chickens on their 42^nd day of life caused a significant (p≤0.01) decrease of the fungi number from 4.2 down to 4.0 log cfu×g^-1 [10]. In the presented experiment, the observed mean values of this parameter were contained in the range from 1.30 log cfu×g^-1 in group IV up to 2.00 log cfu×g^-1 in group II of the birds. Besides, the values did not differ significantly between the investigated bird species within the experimental groups (Fig. 5 and 6).

The observed differences between the mean values of the microorganism number in the intestinal contents, presented in this study and calculated for dietary groups and bird species, have not been confirmed statistically.

The SCFA concentration in the intestinal contents, proving microbiological activity in the birds’ alimentary tract, was negatively dependent (p≤0.01) on the amount of crude fibre, NDF and hemicelluloses in the intestinal contents, and the mean correlation coefficient amounted to – 0.444 (table 4). The pH reaction of the intestinal contents of the examined birds was negatively correlated with SCFA concentration, r = -0.370 (p≤0.05). These results are confirmed by experiments conducted by Marounek et al. [15], who found, during an in vitro experiment, that the amount of SCFA produced in caecum depends to a large range on the type of non-starch polysaccharides. The determined general number of aerobic bacteria and lactic acid bacteria was dependent – to a low and negative but significant (p≤0.05) degree – on the concentration of carbohydrates and pH of the intestinal contents. On the other hand, the number of yeasts and mould was correlated – to a similar degree, but positively (p≤0.01) – with these parameters. The general number of aerobic bacteria and lactic acid bacteria was positively (p≤0.05) dependent on the amount of SCFA. Macfarlane and Macfarlane [14] found that also the bacterial flora composition determines the amount and type of the SCFA produced.

The number of lactic acid bacteria was positively correlated with a general number of aerobic bacteria, and negatively - with the number of yeasts and moulds. A similar antagonistic effect between these microorganisms in the chickens’ caecum contents was found by Jamroz et al. [10]. Bacteria and fungi influence each other. Thomas et al. [24] fund that multiplication of Lactobacillus bacteria is inhibited by yeasts, while Narendranath et al. [17] reduced yeasts development by inoculating different number of Lactobacillus.

CONCLUSIONS

The substitution of ground wheat with ground maize in the concentrates resulted in the increase of xylose contents, and in case of ground barley and oat – additionally the increase of crude fibre, NDF, hemicelluloses and cellulose contents.

The increase of non-starch polysaccharide concentration in the diets increased the general number of aerobic bacteria and lactic acid bacteria in the contents of ileum and caecum of the birds.

A higher number of bacteria was found in ducks in the ileum contents of the groups feding mixtures with the increased contents of structural carbohydrates in comparison with chickens, where no similar tendency was observed.

A higher number of aerobic bacteria and lactic acid bacteria was found in ducks when compared with chickens.

The number of aerobic bacteria and lactic acid bacteria in the intestinal contents was significantly negatively correlated with polysaccharide concentration, while in case of yeasts and moulds – it was positively correlated.

The contents of structural carbohydrates was negatively correlated with SCFA concentration and positively - with pH reaction of the intestinal contents.

A negative dependence between the number of yeasts and moulds and the number of bacteria in the intestinal contents of the birds was found.

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