THE EFFECT OF DIETARY SUPPLEMENT – HUMOBENTOFET ON RABBIT CAECAL PARAMETERS OF MICROBIAL FERMENTATION AT IN VITRO STUDY

Dorota Miśta
Department of Animal Physiology and Biostructure, Faculty of Veterinary Medicine, Wrocław University of Environmental and Life Sciences, Poland

ABSTRACT

The aim of the dissertation was to determine the influence of Humobentofet, a humic-mineral-fatty supplement, on the selected parameters of in vitro fermentation in the rabbit’s caecum. The research was conducted on 54 rabbits of the Belgian giant breed, aged 6 months. The samples of caecal content were taken directly after slaughter and then diluted with a solution containing a buffer selection. To the samples prepared in this way, Humobentofet (HBF) was added at the dose of 10% (group D1) and 15% (group D2) of the taken chyme. The controls consisted of samples that did not contain HBF. The samples were then fermented in vitro in anaerobic conditions in bottles placed in a shaker with a water bath. Collected samples were also prepared and then incubated in a fermentor in order to analyse gases produced in the process. The samples to be analysed were taken from bottles before the fermentation (0. h) and in the course of fermentation, i.e. after 4, 6, and 24 hrs of fermentation, in order to determine the concentration of volatile fatty acids (VFA) in them by means of gas chromatography. Additionally, the pH as well as the concentration of ammonia and lactic acid were also determined. Subsequently, the percentage concentrations of three most important acids (acetic acid C2, propionic acid C3, and butyric acid C4) in their total concentration, the C3/C4 ratio, the fermentation efficiency, the ratio of energy regained in VFA to the energy lost in methane as well as the VFA utilisation coefficient were calculated. The gas samples were taken from the fermentor at 4, 6 and 24 hrs of fermentation to glass pipettes in order to determine the content of methane and carbon dioxide by means of gas chromatography. On the basis of the obtained results, it was concluded that the influence of the humic-mineral-fatty supplement on the in vitro fermentation of the rabbit’s caecal content is manifest first of all in the increase of the content of propionic acid in the total volume of VFA, the increase of the fermentation efficiency and the lowering of the non-glycogenic VFA/glycogenic VFA ratio. Besides, it was observed that the pH of the caecal content was lowered. The changes enumerated above prove the favourable impact of Humobentofet on the bacterial processes that take place in the rabbit’s caecum, especially in the case of animals bred for consumer purposes.

Key words: rabbit, humic-mineral-fatty supplement, caecum, fermentation.

INTRODUCTION

Selected issues of the digestive tract of rabbit

For the last two decades of the last century, a growing interest in the rabbit breeding (Oryctolagus cuniculus L.) has been noticed in many European countries, especially in France, Spain and Italy, thanks to which the rabbit meat has become the equivalent of meat of the other species maintained in intensive breeding [19]. The rabbit meat is distinguished by its high quality and outstanding taste, and it contains more protein and polyunsaturated acids and less fat (below 10 %), cholesterol and sodium compared to red meat and poultry, which are consumed the most often [53].

At the same time, a growing interest in the rabbit as a subject of scientific research has been observed aiming at better understanding of the rabbit physiology and improvement of its feeding and breeding methods. Feeding of the rabbit as a phytophagous animal is more similar to the feeding of ruminants than to the typical animals bred for meat such as pigs or poultry. The rabbit digestive physiology also shows some similarity to the ruminants, especially the caecum processes, which are often compared to fermentation in forestomachs [2, 55, 88]. Apart from that, some characteristics of the rabbit e.g. caecotrophy distinguish digestive physiology of the rabbit from other farm animals. Fermentation, which, with participation of microorganisms, takes place in the rabbit caecum together with the caecotrophy phenomenon enables the rabbit to acquire extra energy, amino acids, and vitamins.

The caecum is crucial for the rabbit digestive physiology as the biggest container for the fibre degradation and fermentation processes. It is rich in bacterial flora with predominant completely anaerobic bacteria for which carbohydrate fermentation is the main source of energy. The carbohydrates that get into the caecum are mostly non-starch polysaccharides and little starch (neutral detergent fibre – NDF constitutes 27.7 to 36.1 % of dry matter of the caecum contents, whereas starch – 0.9 to 1.6 %) [55,71].

Fermentation in the rabbit caecum resembles metabolism in the rumen of the ruminants, however there are certain features which distinguish one process from the other. A characteristic of the caecum fermentation in young rabbits is acetogenesis (reductive acetogenesis) dependent on hydrogen, which, with age, is partly replaced with methanogenesis [70]. The reductive acetogenesis is the synthesis of acetate from CO₂ and H₂ with participation of microorganisms. The same metabolism also takes place in human colon and pig’s caecum but is not limited to them. The reductive acetogenesis is of relatively little importance in the ruminant forestomachs except for a short period following birth [64]. It is also considered to be an alternative way of metabolism for methanogenesis in the rumen as the reductive acetogenesis bacteria were also found in the rumen of adult animals. Proportions of the produced volatile fatty acids are another feature that distinguishes the processes in the rabbit caecum from the rumen fermentation. Unlike the ruminants, butyrate concentration is higher than propionate concentration in the rabbit caecum. The differences between the caecum and the rumen may be reduced through fasting rabbits, which as a result of the lack of substrates, inhibits the growth of bacteria that participate in the reductive acetogenesis [55].

Being a more perfect form of coprophagy, which is widely present in rodents, the phenomenon of caecotrophy observed in the Lagomorphs plays an important role in the use of both the products of the microorganism fermentation and the amino acids and vitamins produced by the microorganisms. It consists in isolation of the large intestine contents particles which are less digestible or nutritive and their excretion in the form of hard faeces, whereas most bacteria, fluid and small caecum contents particles are saved thanks to retrograde motion in the proximal colon. The so-called soft faeces enveloped in mucus are eaten straight from anus [12,15,82]. Compared to hard faeces, which contain more fibrous components, the soft faeces contain more proteins, including essential amino acids such as lysine, sulphuric amino acids, threonine [13,79], mineral components, and vitamin K and B group. The soft faeces also contain enzymes, including the fibrolytic ones [47]. According to Piattoni et al [71], approx. 50 % of nitrogen in the caecum comes from bacteria source. It is recovered thanks to the caecotrophy, thus 12 – 24 % of nitrogen supplied to the rabbit organism comes from the caecal microorganisms [41].

The microflora that inhabits the caecum is constituted mainly by the Bacteroides genus bacteria in the number of 10⁹ – 10¹⁰ bacteria/1g of contents [13,45], and Bifidobacterium sp., Clostridium sp., Streptococcus sp., Enterobacter sp. complete the population giving the total number of 10¹⁰ – 10¹² bacteria/1g [8,13,30,44]. A lack of the Lactobacillus genus bacteria is a characteristic feature of the rabbit intestine environment [32]. The following various bacterial activities have been found in the caecum: fibrolytic (cellulolytic, pectinolytic and xylanolytic) [10], ureolytic [31], proteolytic [25], amylolytic [68], as well as, from the 36th day of the rabbit life, a methanogenic activity [70]. Compared to the rumen of the ruminants, the fibrolytic activity in the rabbit caecum is lower, whereas the amylolytic activity and proteolytic activity are relatively higher, due to the host’s enzymes getting in here [41]. The pectinolytic activity in the rabbit caecum rises with its age and, in time, it is a few times higher than the xylanolytic and cellulolytic activity [39,58]. The research also showed a higher number of the pectinolytic and hemicellulolytic bacteria population in the intestine contents than the number of the cellulolytic bacteria [10].

Implantation of the cellulolytic flora begins as early as at about the 3rd week of age when the rabbit starts to take solid food. The presence of cellulolytic flora is associated with the production of volatile fatty acids (VFA), mainly acetic, propionic and butyric acids, whereas the amylolytic flora contributes to the production of the branched fatty acids and valeric acid [68]. The production of volatile fatty acids grows simultaneously with the growth of the cellulolytic bacteria number and, between the 15th and 25th day of life, the acid concentration increases approx. four times, and, at the same time, the proportion of the propionic acid to the butyric acid gets reversed [43,68,70,71,81].

As a result of the presence of microflora, the intestines are a highly immunogenic environment, therefore the intestine regulation of the immunological answer is necessary. The volatile fatty acids participate in it with their anti-inflammatory reaction through, among other things, leukocyte activity modulation [14,74]. An inhibiting impact of the acids on multiplication of the pathogenic bacteria in the large intestine is also known [11]. An influence of the volatile fatty acids was also found on the digestive tract motility [16] and the intestine secretion, and coordination of the two mechanisms enables the participation of the volatile fatty acids in the integration of the large intestine functions [84].

Concentration of the final products of the bacterial fermentation in the digestive tract, especially volatile fatty acids and ammonia, reflects the activity of microflora, which conditions a proper course of the processes that proceed here [32]. Changes in feeding may modify the fermented contents of the caecum, and consequently, the activity of micoorganisms and stability of the bacterial flora.

The humic-mineral-fatty preparation in rabbit feeding

The application of additives in animal feeding, which are the products of chemical synthesis or biotechnological processes, is often the subject of criticism from the doctors, nutritionists, and ecologists, mainly due to a negative effect of the preparations on stability of the microflora of the digestive tract and the penetration of the harmful substances to the animal tissues and organs, which are then consumed by people. This is why the research on the use of natural raw materials and organic and mineral products is often undertaken, which improve the breeding parameters without the above-mentioned side effects.

The research on the use of the biological properties of the humic compounds contained in peat and brown coal has been carried out lately. Humobentofet (HBF) is one of such preparations which are vegetable oil mixture on humic-mineral carrier. The research on Humobentofet carried out so far has proved its feed preservative features and favourable effects on feeding of fattening pigs, sows, cattle, sheep, and poultry. The rich composition of Humobentofet enables supplementation of energy and mineral deficiencies in substantial mixtures for animals, has a favourable effect on animal health and productivity, improves nutrient digestibility, increases body weight gain and decreases feed consumption per 1 kg of weight gain. As the application of the above-mentioned preparation as well as other additives which contain humic compounds has already been positively assessed in the case of pig, poultry, cattle and sheep feeding [21,22,23,48,49,50,60,69,75,76], the application of Humobentofet seems to be also interesting for the rabbit feeding.

According to its manufacturer’s information, Humobentofet shows strong bactericidal and antifungal action and absorbs toxins produced by the microorganisms. It reduces acidity in the digestive tract and absorbs up to 70 % of gases produced in it (ammonia, hydrogen sulphide, mercaptans). Besides, the humic-mineral preparations prevent adverse decay processes in large intestine, whereas most of acidifiers and antibiotic additives is already absorbed in the small intestine and does not act in further sections of the digestive tract. Silica contained in Humobentofet causes slowing down of chyme movement in the intestines which results in a 30-per cent increase in cellulose digestibility through which the use of mineral compounds contained in seed husks increases.

The main ingredients of Humobentofet are the humic compounds (humodetrynit) and fatty acids based on bentonite: oleic, linoleic, linolenic, palmitic and other. The mineral composition of the preparation includes, among other ingredients, the most important macro- and microelements which should be included also for the rabbit feeding such as calcium, phosphorus, magnesium, sodium, potassium, iron, copper, manganese, zinc, selenium, and cobalt [22].

Even the most varied feeding doses used for rabbits should be enriched with mineral ingredients and vitamins [66]. Ca, P, Mg, Na, K, Cl and S are the most important among the macroelements for the rabbit feeding, whereas Fe, Cu, Mn, Zn, Se, I and Co are distinguished among the microelements, which are considered to be necessary for the rabbit feeding. During pregnancy and lactation, attention should be drawn to enrichment of feed with calcium because doe milk is very rich in this chemical element. It is also important to supplement the feed of the pregnant does with iron. The mechanisms of Fe transport to milk are poor in most of mammals, however the does can protect their litter by transferring a considerable amount of iron through placenta [59].

Both the macro- and microelements affect the processes that proceed in the animal’s organism in the different way and, through their presence in the digestive tract, they also influence the fermentation processes in here. An impact of the main mineral ingredients on the rumen contents fermentation had been found consisting in the regulation of its physicochemical properties such as osmotic pressure, buffering ability or oxido-reduction potential. Besides, some chemical elements may directly influence the course of the bacterial processes. Thus, for example, cellulose degradation in suspension of rumen bacteria cells is accelerated by the following elements: P, Mg, Ca, K, Na, Fe, Mn, Zn, Co, Mo, whereas the volatile fatty acid synthesis in the rumen fluid may be increased by Mn, Co and Zn [24].

However, an application of fat additive in the rabbit feed is the subject of a lot of research. Both vegetable and animal fat supplied in feed for the rabbits is well used as a condensed source of energy [66]. According to some authors, inclusion of fat in the rabbit feed causes reduction of its consumption per 1 kg of body weight gain [28,29]. According to Pote et al [72], the inclusion of fat in the rabbit feed may be the way of maintaining of such a level of energy as in the high-energy feed, yet without loading the large intestine with carbohydrates. In addition, it has been found that vegetable fat increases a content of digestible energy in rabbits without any negative impact on digestibility of fibre contained in feed as, for instance, in the case of ruminants [28,77].

An increase in the body’s resistance is also an important effect of the supply of fat in feed. An impact of lipides on the immune response, both on a level of intestines and a systemic level, is probably the best-documented relation between feeding and immunity. The greatest immunomodulation impact has been found in the case of polyunsaturated acids (PUFA) ω3 and ω6. Arachidonic acid, the main product of their metabolism and precursor of leucotrienes, influences production of LT4 and PGE₂, which induce a number of favourable immune reactions [32].

A favourable effect of the unsaturated fatty acids provided with food on the rabbit’s organism as well as the rabbits’ demand for mineral ingredients induce to make an attempt to apply the mineral-fatty preparation for the purpose of the rabbit feeding. In connection with the above, examination of an impact of Humobentofet on the course of the fermentation processes in the digestive tract of the rabbit seems to be an interesting research issue.

The research has aimed at determination of an impact of HBF, a humic-mineral-fatty feed additive, on the in vitro fermentation parameters in the rabbit’s caecum, i.e.: chyme reaction, contents and mutual proportions of the volatile fatty acids, lactic acid and ammonia concentration, contents and mutual proportions of gases produced i.e. carbon dioxide and methane, fermentation efficiency, ratio of non-glycogenic to glycogenic volatile fatty acids.

Examination of the factors that change fermentation profile with the use of the in vitro method enables measurement of metabolites, which, in in vivo conditions, are quickly absorbed by the digestive tract wall, eliminates the host’s reactions to the local microflora disturbance, and enables observation of dynamics of the changes in chyme on the basis of measurements performed at short intervals of time. The changeability observed between fermenters is similar with the one observed in vivo between the animals, and in addition, the experiments carried out with the use of the in vitro method enable elimination of the individual differences in feed intake and working of the digestive tract [1]. Yet, as a result of different conditions of the in vitro experiments conducted by various authors, the best solution is to treat a given fermenter as one’s own reference point [2].

MATERIALS AND METHODS

The research was conducted on 54 rabbits of the Belgian giant breed, aged 6 months with a weight of 4.5 kg approx., fed with FK/W granular mixture (manufactured by Dolpasz S.A.) in accordance with feeding standards [3]. The animals received water and feed ad libitum.

For conducting of the fermentation in the stomach and caecum contents, the in vitro method was used, which enabled observation of the course of the processes with determination of 4 measurement times (after 0., 4., 6. and 24. hours of the fermentation).

The contents of caecum was taken directly after slaughter and then samples for the research purposes were prepared, which included 10 g of chyme and 90 ml of a solution containing buffering compounds properly selected [2]. Such fluid with a reaction corresponding with a reaction of ileum fluid of the rabbit (about 7.5), should ensure a buffer ability of the environment and provide necessary ingredients for the growth of bacterial flora.

To the samples prepared in the said way, Humobentofet (HBF), a mineral-fatty preparation was added, which was manufactured by the company PHW “Tronina” and is registered in MRiGŻ as a feed additive (ref. No. RRpp-4121-C-350/97). Chemical composition of Humobentofet has been shown in Table 1. An energy value of Humobentofet amounts to 8MJ (1912 kcal) kg^-1 [22].

For the experimental purposes, 3 groups have been created: K – control samples without the HBF additive, D₁ – samples in which HBF constituted 10 % of the chyme taken (prior to dilution), and D₂ – samples in which HBF constituted 15 % of the chyme. All samples were subject to the in vitro fermentation in glass bottles with a capacity of 300 ml adjusted specifically for this purpose and placed in a shaker with a water bath [86, 87].

Collective samples prepared in a similar way were subject to incubation in a fermenter with a capacity of 1600 ml (140 g of the chyme and 1260 ml of the buffer) in order to analyse gases: carbon dioxide and methane produced in the process. A greater capacity of the fermenter ensured receiving of higher concentration of the gasses produced and facilitated measurement even of relatively small concentrations of methane. The dilution method was identical with the previous one, whereas the samples were divided only into two groups: K – control samples without the Humobentofet supplement and D₁ – samples in which Humobentofet constituted 10 % of the chyme taken.

Both the samples in bottles and the ones in the fermenter were flashed with nitrogen prior to the fermentation in order to achieve the anaerobic conditions, which were controlled by means of an oxygen meter DO-5510 manufactured by LUTRON. The incubation was carried out at a temperature of 39°C.

The samples of chyme for analysis were taken from the washer prior to the fermentation (0. h), and also in the course of the fermentation i.e. after 4, 6 and 24 hours, in order to determine the concentration of volatile fatty acids (VFA) in them: acetic acid (C₂), propionic acid (C₃), isobutyric acid (iC₄), butyric acid (C₄), isovaleric acid (iC₅) and valeric acid (C₅), by means of gas chromatography. Additionally, the concentration of ammonia and lactic acid as well as the pH value were also determined in the examined samples. Analyses of the ammonia and lactic acid concentration were carried out based on the calorimetric methods and the results were given in mmol·l^-1. The level of ammonia was determined with the use of the Conway’s method, whereas the content of lactic acid was measured with the use of the titration method. The pH measurements were performed by means of a pH-meter CP-401 manufactured by ELMETRON with an electrode EPP-3 and a temperature sensor. Prior to each series of the measurements, calibration of the electrode indications in three standard solutions was performed (pH 4, 7 and 9).

For determination of the content of the volatile fatty acids, a gas chromatograph series 104 manufactured by PYE UNICAM was used with a detector FID, which is universal to organic compounds. The chromatograph was equipped with a capillary column manufactured by Supelco with a length of 2.1 m, whose packing consisted of a solid phase – Chromosorb W AW, with granulation 80/100 and a liquid phase – 10 % of carrier (SP 1200 + 1% H₂PO₄). Nitrogen was a carrier gas fed from a gas bottle. Velocity of the mobile phase flow through the column amounted to 60 ml min^-1. The chromatographic analyses were performed in an isotherm 135°C at a feeder temperature of 150°C and a detector temperature of 200°C. A volume of injection amounted to 1 μl. Calibration curves were determined on the basis of an analysis of the proper dilution of the standard solution of the volatile fatty acids provided by the Supelco. The chromatograms were registered by means of an Acord computer system. The results were provided in mmol·kg^-1 of chyme.

After the results of concentration of the particular volatile fatty acids had been received, the fermentation parameters were calculated such as percentage of molar concentrations of the three most important acids i.e. acetic, propionic, and butyric acid in their total concentration, propionate – butyrate molar concentrations ratio, fermentation efficiency, and VFA utilization coefficient.

The fermentation efficiency was calculated on the basis of the equations worked out by Czerkawski for the ruminants [20] and modified by Baran and Zitňan [4]:

E₁ = energy contained in VFA /energy in fermented hexose = [62 + 0.47(p + 2b + 3v)] 100/(100 + b + v)

E₂ = energy contained in methane/ energy in fermented hexose = [28 – 0.47(p + v)] 100/(100 + b + v)

where: a, p, b and v express percentage of the molar concentrations of the acetic, propionic, butyric and valeric acid, respectively, in the total concentration of the VFA examined; E₁ means recovered energy in VFA as a percentage participation of output energy, whereas E₂ shows a level of lost energy in the methane also expressed in percentage. The fermentation efficiency is expressed by an E₁ : E₂ ratio.

For the comparative purposes, another equation for the fermentation efficiency was also applied worked out by Ørskov [67] and modified also by Baran and Zitňan [4]:

FE = (0.622a+1.092p+1.56b) 100/(a+p+2b) (notations as mentioned above).

The final result of this equation is also expressed in percentage and shows an amount of energy stored in VFA as a percentage participation of the initial energy.

The VFA utilization coefficient has been expressed by non-glycogenic VFA/glycogenic VFA ratio according to Ørskov [67]:

NGGR = (A + 2B + V)/(P+V)

where A, P, B and V express the concentrations (mmol·kg^-1) of the acetic acid, propionic acid, butyric acid and valeric acid, respectively.

NGGR is expressed by a ratio of absorbed substrates which can transform neither into glucose nor any of its precursors to the substrates which can transform into glucose or participate in glucose transformation. In the case of volatile fatty acids, NGGR shows a ratio of non-glycogenic VFA to glycogenic VFA and indicates their utilization. The valeric acid is classified under both above-mentioned groups because, as a result of its oxidation, 1 mole of the acetic acid and 1 mole of the propionic acid are created. Too high a level of NGGR indicates high loss of energy (in the form of gases).

Samples of gases were taken from the fermenter after 4., 6. and 24. hours of fermentation into glass pipettes with a capacity of 250 ml equipped with two tight taps in order to determine the content of methane and carbon dioxide. For standardization of the measurement conditions, nitrogen was introduced each time to replace the drawn gas, and as incubation gas, nitrogen was then used to fill the fermenter thanks to which each measurement regarded the gas production from the moment of the previous sample collection. The results were given in percentage of volume of gas contained in a sample. Determination was performed by means of a chromatographic method with the use of two N504 gas chromatographs manufactured by Elwro with detectors TCD and FID. A chromatograph with the TCD detector was equipped with a column with a diameter of 4 mm and a length of 1.9 m, filled with Carbosiev B 80/100 mesh manufactured by Supelco, Inc. Helium was a carrier gas, flow velocity amounted to 40 ml min^-1, a column temperature – 80°C (isotherm), a temperature of a feeder and detector –150°C. A chromatograph with the FID detector was equipped with a column with a diameter of 4 mmm and a length of 2.5 m, filled with Poropak Q+R, 80/100 mesh, manufactured by Supelco, Inc. Nitrogen was a carrier gas, flow velocity amounted to 35 ml min^-1. An initial temperature amounted to 60°C (3 min), linear accretion 8°C/min to 220°C (5 min), a temperature of a feeder and detector was 250°C.

The results of the experiments were subject to a statistical analysis with the use of a model of a two-way variance analysis with interactions. Apart from significance of an impact of individual effects (time and the addition of HBF), significance of their mutual co-operation or interaction was also examined allowing to assess an influence of the fermentation time within each experimental group compared to the control group. Significance of differences was estimated by means of the Tukey’s multiple comparison test. Differences were considered to be significant at P<0.05. An assumption that variances are equal in each subgroup was verified with the use of the Levene's test. All calculations were carried out in the Statistics Unit of the Mathematics Department of the University of Environmental and Life Sciences with the use of statistic package STATISTICA ver. 6.2.

RESULTS

Value of the reaction of the caecum contents which underwent fermentation decreased considerably in the course of the fermentation (Table 2). The differences among the groups with the added Humobentofet were also observed compared to the control group (Fig. 1). After the 24-hour fermentation pH in the control group decreased from 7.5 to 7.16, in D₁ – from 7.32 to 7.02, and in D₂ – from 7.4 to 6.98.

In the case of the microbiological metabolism products, and significant impact of time on their concentration in chyme was always noticed, which increased between 0. and 24. hour of the fermentation (at p ≤ 0.05).

The total concentration of volatile fatty acids in the caecum contents before fermentation amounted to approx. 72.57 mmol·kg^-1, and no significant differences influenced by the added Humobentofet were noticed between the groups during the fermentation (Table 2).

Concentration of the acetate (C₂), which occurred in the digestive tract contents of the rabbit in the greatest amount, did not change considerably during the fermentation under the influence of HBF. However, a small drop in percentage of the acid in groups D₁ and D₂ was observed (Table 3). Thus, prior to the fermentation, the percentage amounted to 62.1 % in group K, 62.04 % in D₁ and 62.0 % in group D₂, and after 24 hours it reached the values of 61.74, 57.33 and 56.63, respectively.

Among the volatile fatty acids in the examined section of the digestive tract, the greatest changes in the content of the propionate (C₃) were observed. An increase in the concentration of the propionate under an impact of the added HBF was proved, as well as a result of interaction of both factors examined i.e. the additive and the fermentation time (p ≤ 0.05). The greatest differences occurred between groups K and D₂ after 6 and 24 hours of the fermentation (Fig. 2, Table 2).

Simultaneously with the increase of the propionate concentration influenced by the added HBF, its percentage in the total amount of the three most important volatile fatty acids i.e. acetic acid, propionic acid, and butyric acid increased, too (Fig. 3, Table 3).

No significant difference in the concentration of the butyrate (C₄) caused by the influence of the examined humic-mineral-fatty additive was found. The percentage of C₄ in the caecal contents also did not change, either and oscillated at a level of 26.82 approximately (Tables 2 and 3).

Considering the percentage of molar concentrations of the acetate, propionate, and butyrate in their total amount, it should be stated that, with time, the percentage of the examined acids did not change considerably in the control group, whereas the HBF supplement caused a decrease in the percentage of acetate and increase in the percentage of propionate compared to the control group (Fig. 4, Table 3).

The content of acids which occur in a smaller amount in the chyme (iC₄, iC₅, C₅) was analyzed as a total concentration (as min VFA = iC₄ + iC₅ + C₅), as well as separately for each acid. An impact of Humobentofet on a decrease in the concentration of min VFA was found (Fig. 5, Table 4).

Similar changes were observed in the case of concentrations C₅, iC₄ and iC₅ analyzed separately, whose level in group D₂ had the lowest values and differed significantly from the remaining groups (Fig. 6, 7 and 8, Table 4). Isobutyric and isovaleric acids occur in traces in the rabbit caecum and, in this research, the concentration of the acids, just after the chyme had been taken, amounted to 0.31 and 0.47 mmol·kg^-1, respectively.

While analyzing the remaining products of the microorganism metabolism in the caecum, an significant impact of the additive examined on a level of ammonia in the fermented chyme was found. Molar concentration of ammonia in the examined material grew during the fermentation in all experimental groups, and the greatest rise was noticed in group D₂, and then group D₁ (Fig. 9, Table 3).

An impact of the HBF supplement proved to be significant in the case of an analysis of concentration of lactic acid in the caecum. The humic-mineral-fatty additive contributed to a faster increase in a level of the above-mentioned acid during the fermentation in groups D₁ and D₂ compared to the control group (Fig. 10, Table 3).

Among the fermentation parameters calculated on the basis of concentrations of the relevant products of the microorganism metabolism, ratio C₃/C₄, the fermentation efficiency, and the use index NGGR have been analyzed in this paper. The value of C₃/C₄ did not change significant during the fermentation of the caecum chyme, yet, in particular time intervals, it was slightly higher for the groups with added HBF (Table 4). After 0 hours, a ratio of concentrations of the above-mentioned acids amounted to approx. 0.48, whereas after 24 hours it was close to the initial value in the control group, it amounted to 0.66 in D₁ and 0.68 in D₂.

Fermentation efficiency (FE) calculated on the basis of concentrations of volatile fatty acids determined in the caecum contents increased under an influence of the added HBF (Fig. 11), reaching the greatest value in groups D₂ and D₁ after 24 hours of fermentation, whereas it did not undergo any significant changes in group K at the same time (Table 5).

Similarly to FE, an impact of time and Humobentofet (p≤0.05) on an increase in the value of E₁ was noticed, which, yet, reached the highest value in group D₁ after 4 and 6 hours of fermentation (Fig. 12, Table 5).

The value of E₂ which indicates the content of energy lost in methane was approximately inversely proportional to E₁, i.e. decreased in groups D₁ and D₂ in the course of fermentation, and the HBF supplement significantly influenced a decrease of the value of E₂ in the above-mentioned groups compared to the control group (Fig. 13). The greatest differences regarded the 24th hour of fermentation (Table 5).

Ratio of E₁/E₂, which indicates efficiency of processes that take place in chyme, reached an average value of 5.07 in the caecal contents (Table 5), in addition to which an significant influence of the fermentation time (p≤0.05) and Humobentofet on the ratio was observed (Fig. 14). In the course of 24-hour fermentation, the E₁/E₂ ratio increased from 4.65 to 5.51 in group D₁, and from 4.44 to 5.54 in group D₂, whereas it remained at the similar level in the control group (4.8 and 4.72, respectively). The greatest values were obtained at the 4th hour of fermentation for group D₁ and at the 24th hour for groups D₁ and D₂.

In the case of NGGR, which indicates the ratio of non-glycogenic to glycogenic volatile fatty acids created as a result of the fermentation, the coefficient showed significant differences influenced by the fermentation time (p≤0.05) and under the influence of both: time and the HBF supplement (p≤0.05)(Fig. 15, Table 5). In the control group, an increase of the NGGR value was noticed at first, and then, its slight decrease NGGR (6.07 at 0 hour, 7.41 after 4 hours, 7.26 after 6 hours and 6.68 after 24 hours), whereas a decrease of the value was observed in groups D₁ and D₂ (from 7.87 and 8.18 at 0 h to 5.20 and 5.09 after 24 hours, respectively).

Summing up of the analysis of the fermentation parameters calculated on the basis of VFA concentrations, it can be stated that, thanks to the change of the fermentation profile obtained with the use of the humic-mineral-fatty supplement, efficiency of the fermentation in the caecal contents increased, the percentage of energy stored in the volatile fatty acids grew, the content of energy lost in methane decreased, and the ratio of non-glycogenic to glycogenic glikogennych volatile fatty acids decreased in the course of fermentation.

The analysis of gases produced as a result of activity of the intestine microflora showed only an influence of the fermentation time on the percentage concentration of methane in the total volume of gases in a sample examined (p≤0.05, Table 6). In spite of the lack of the statistically significant differences, samples of the gas that came from the content which included Humobentofet, mostly showed greater average production of CH₄ compared to the control group.

The content of carbon dioxide produced in the course of fermentation was a few times larger than the content of methane which is reflected in the calculated ratio of CO₂/CH₄ (Table 6). In the course of fermentation, an influence of time and Humobentofet on an increase in the content of CO₂ in the analyzed gas samples was noticed (p≤0.05), which proves an increase in intensity of the fermentation processes. No significant differences related to the ratio of CO₂/CH₄ influenced by HBF were found, however, its values were higher for the samples with the supplement taken after 4 and 6 hours of fermentation compared to the control group. This could indicate relatively lower production of methane in the samples with the humic-mineral-fatty supplement, despite the percentage participation of this gas was even slightly higher than in the control samples. After 24 hours of fermentation, the CO₂/CH₄ ratio reached a similar value in both groups (approx. 2.48).

DISCUSSION

Digestive disorders in the rabbit are almost always associated with upsetting the balance of proportions of the produced volatile fatty acids, thus it is necessary to monitor their content continuously. Inflammatory condition and parasitic invasion in the digestive tract may also be the reason for disturbance of the production of these compounds [54,62]. Some changes in the total concentration and proportions among the particular volatile fatty acids influenced by the factors associated with ontogeny and feed composition are also observed.

In this paper, the total concentration of the examined volatile fatty acids in the rabbit caecum amounted to 72.57 mmol·kg^-1 before fermentation (0 h). According to Garcia et al [36], the research that has been carried out in three leading centres in Europe for the last years (UPM – Madrid, INRA – Tulouse, UTL – Lisbon) and regarded the products of the bacterial fermentation in the caecum contents of rabbit, has shown that the total level of volatile fatty acids could reach the value from 18.1 to 99.8 mmol·l^-1, depending on the rabbit’s age and physiological status as well as food ingredients. Analysis of the above-mentioned data also showed variation in proportions of the molar concentrations of the following acids: acetic, propionic and butyric. The percentage participation of acetate amounted from 64.7 to 87.2, propionate – from 3.3 to 11.1, and butyrate 5.76 – 28.4. In this paper, the proportions amounted to 62.04 : 11.98 : 25.98, respectively after 0 h of fermentation. The rabbit’s age accounts for slightly smaller participation of acetate and greater participation of propionate in the caecum contents as the animals aged up to 3 months (with a weight 1 – 2 kg) have been mostly used for the experimental purposes in the above-mentioned centres, whereas for these experiments, 6-month old rabbits have been used (approx. 4.5 kg). The previous research proves that, with age, participation of acetate in the total amount of VFA decreases and the participation of propionate and butyrate increases [6,47].

The content of VFA in the rabbit caecum is also influenced by the time when the contents was taken depending on feeding time and time of the day, which is associated with caecotrophy. According to Gidenne and Bellier [38], concentration of the above-mentioned volatile fatty acids in this intestine increases four times after feeding and reaches the maximum value at the fermentation peak i.e. 5 hours after feeding, in addition to which participation of butyrate in the total amount of VFA grows and the ratio of propionate to butyrate changes to characteristic for the species. In the case of measurement carried out before feeding, the C₃/C₄ ratio gets inverted. The influence of the feeding time may be reduced by giving food to animals ad libitum, as in the case of this research. With this way of feeding, there is still an impact of time of the day left at which samples of the intestine contents were taken, considered by Garcia et al [36] as one of the more important reasons for different results regarding concentration of the fermentation products. Before and during the phase of soft faeces excretion, the content of VFA in the caecum chyme was noticed at a level below 20% of their total amount produced in during hard faeces production [40,41]. The reason for this phenomenon is sought in an increase of food taking in the phase of hard faeces and enrichment of the caecum contents with populations of micoorganisms during antiperistaltic movement of proximal colon which take place at this time [5,13].

During the in vitro fermentation, which occurs in closed systems, accumulation of the products of bacterial activity in the fermented chyme takes place [2,80]. During this research, an increase in molar concentrations of most of the volatile fatty acids investigated was observed during the in vitro fermentation, yet their proportions also changed. In order to enable confrontation with the data available in the literature, percentage participation of concentrations of the three most important acids was compared to the total of their concentrations in the chyme [6,35,39,71].

Under the influence of the added Humobentofet, an increase of participation of the propionate compared to the control group was observed in this research. This acid, apart from the acetate, is one of the crucial sources of energy for hepatocytes. An activating impact of propionate in the colon lumen on mucosal Cl^- secretion in this intestine was found [84]. The propionate also intensifies absorption of iron in the proximal colon [9]. It is also a very effective substrate in glucose synthesis in many species [7], it participates in glycogenosis and formation of long-chain fatty acids in the liver and intermediate products of its changes take part in regulation of a series of processes, including ketogenesis, gluconeogenesis, ureogenesis, or β-oxidation [73]. Thus, the increase in production of the propionate in the digestive tract seems to be favourable, especially for the animals reared for the consumption purposes.

In this research, the increase of participation of propionate in the total amount of volatile fatty acids took place, first of all, with decrease in the participation of acetate. The acetate participates in lipogenesis (in the human and bird liver, in fatty tissue of the ruminants and pigs, and in both above-mentioned tissues in rodents), in milk fat synthesis, cholesterogenesis and ketogenesis, and it can also activate gluconeogenesis from lactate and pyruvate [73]. There are reports about endogenous production of the acetate outside the intestine tissues (from non-estrified fatty acids), as a result of which the total amount of acetate in circulating blood prevails over the total amount of acetate absorbed from the digestive tract [7,51].

Among the volatile fatty acids investigated in this research, the smallest changes, resulting from the addition of the humic-mineral-fatty supplement in production of butyrate were observed, especially in the caecum. Butyrate is the basic metabolic ‘fuel’ in the large intestine tissues of the rabbit and in the rumen wall of the ruminants. A low level of the butyrate in the caecum may be a reason for inflammatory processes of intestines in rabbits [15]. It was found that, the n-butyrate and, to a lesser degree, the propionate inhibit a growth of a line of colon carcinoma cells through induction of their apoptosis [17,26,78]. Additionally, the acid may be a precursor in lipogenesis and ketogenesis, and it also serves as an activator of gluconeogenesis from lactate and ureogenesis, yet it is likely to inhibit gluconeogenesis from propionate [26,73].

An increase in participation of the propionate in proportion to the acetic and butyrates in the caecum was also obtained, under an influence of fat added, by Falcão-e-Cunha et al [27] in an in vivo experiment, in which the rabbits received sunflower oil in feed in an amount of 60 g·kg^-1 as a substitute of corn starch. The above-mentioned supplement still lowered the total level of volatile fatty acids from 43.7 to 39.9 mmol·l^-1, in addition to which it should be stressed that both effects regarded the rabbits fed with the feed which contained wheat bran, whereas such an influence of fat addition was not observed in the case of rabbits fed with the feed with dehydrated lucerne (bulky feed).

During fermentation in a sheep’s rumen, an influence of the cis-oleic acid and linolenic acid added to feed also manifested itself with a decrease in participation of the acetate and an increase in the propionate with small differences in a percentage content of the butyrate, thus similarly to the influence of HBF in the rabbit caecum in this research, whereas the linolenic acid also influenced an increase in a level of C₄ apart from reducing the participation of C₂ and increasing C₃ [85].

An influence of bentonite (which also occurs in the composition of HBF) on the parameters of the rumen fermentation in sheep was also examined, and no significant differences were found in the case of animals fed mainly with bulky feed, whereas when they were fed with concentrate, an increase in the percentage participation of acetate and a decrease in the content of propionate were obtained (mol%) [65], thus the effect was opposite to the one obtained in this research. Galyean and Chabot [34] did not find any significant changes in the concentrations of volatile fatty acids and ammonia in the bull rumen under an influence of the mineral compounds added to feed (including sodium bentonite). Addition of magnesium-mica (magnesium-potassium silicate) to the heifer feed (2.5, 6.25 and 10 %) did not cause either any significant changes in the content of VFA in the rumen [18]. The above-mentioned data seems to indicate that the changes in the VFA profile obtained under the influence of HBF are, first of all, a result of an impact of the unsaturated fatty acids contained in the preparation.

Metabolism of the volatile fatty acids is less efficient than metabolism of glucose or long-chain triglycerides, as more energy is used for formation of mole of ATP [51]. In this research, the fermentation efficiency was calculated based on converting of the hexose energy to the VFA energy on the basis of equations for the rumen fermentation and heat of combustion of hexose (glucose), acids: acetic, propionic and butyric, and methane [4,67]. The values calculated regard energy from the fermented hexose bounded in the volatile fatty acids (E₁) and methane (E₂) and like FE, they are based on the percentage participation of the individual LKT in their total amount. As methanogenesis is partly replaced with reductive acetogenesis in the rabbit caecum and the creation of volatile fatty acids slightly differs from that in the rumen [52,83], the values of energy calculated in percentages do not reflect accurately the energetic changes in rabbit, however it is possible to estimate comparatively the fermentation efficiency on the basis of the said values for the experimental groups.

In this research, an increase in the FE fermentation efficiency compared to the control groups influenced by the humic-mineral-fatty supplement was observed. E₁ calculated for all experimental groups oscillated at a similar level as FE, whereas a content of energy lost in methane decreased under the influence of the HBF supplement. E₂ indicates a percentage participation of the energy contained in methane in the total amount of the energy obtained, therefore this value does not have to be correlated with a percentage participation of CH₄ in gas samples taken. The volume of methane and carbon dioxide will grow with intensive fermentation, and the production of VFA will increase simultaneously.

Efficiency of the fermentation in the rumen of the sheep fed with forage with added 50 % of concentrate (FE = 76.9 % in the fresh chyme taken) [85] oscillated at a slightly higher level than the one obtained in this research. A lower value of FE, close to the one obtained at 0. h of the fermentation in this research (73 %), was achieved in the sheep fed with bulky feed (72.84 %). Under an influence of the non-saturated acids: cis-oleic, linoleic and linolenic (which are also contained in Humobentofet) added to feed, an increase in the fermentation efficiency occurred in the rumen of both the sheep fed solely with forage (up to 76.6, 76.1 and 76.0 %, respectively) and the sheep that received 50 % of the concentrate in its nutritional doses (82.9, 83.0 and 83.7 %, respectively).

An increase of the rumen FE in sheep as well as an increase of E₁ and decrease of E₂ was also achieved during the fermentation after the application of methanogenesis inhibitors such as monensin [4]. These results regarded the animals fed with bulky fodder and concentrate in various proportions (the greatest participation of the nutritive feed amounted to 50 %), and an increase of the values of FE and E₁ was associated with an increase in the molar concentration of propionate and decrease of acetate and butyrate in the rumen chyme.

The values of the volatile fatty acid utilization index (NGGR) proved to be higher for the caecum of a rabbit than the rumen of ruminants in which an optimum value of the index amounts to 3.5 and its higher values indicate the worse use of VFA [20]. Zawadzki [85] obtained a slightly higher value of NGGR solely in the nursing sheep with the young, in which the index amounted to 4.03. In this research, the above-mentioned values fell during the fermentation under the influence of the applied humic-mineral-fatty supplement, which indicates an increase in the participation of the glycogenic VFA in their total amount. In the control group, the value of NGGR remained at the similar level in the course of fermentation. The lowest value of NGGR, which indicates the best utilization of VFA, was obtained under the influence of the added 15 % of HBF supplement (5.09).

Improvement in the VFA utilization under the influence of the non-saturated fatty acids added to feed was also obtained by Zawadzki [85] in the sheep rumen in an in vivo experiment where addition of the cis-oleic acid , the linolic acid and linolenic acid reduced the value of the NGGR index.

Environment of the large intestine is considered to be strongly proteolytic thanks to the presence of the population of amino acid fermenting bacteria. As a result of activity of the bacteria, ammonia, CO₂ and, depending on a substrate, various VFA are created, including acetic, butyric, propionic, isobutyric, 2-methylobutyric or isovaleric acid. A series of factors influence the amino acid fermentation and hence the production of ammonia, including H₂ pressure, chyme reaction (which directs the changes towards deamination or decarboxylation), presence of carbohydrates (for example, glucose inhibits activity of some proteolytic bacteria), and, in the in vivo conditions, a time in which the chyme remains in the intestine [52].

In this research, the ammonia concentration in the fresh chyme taken from the caecum (0. h) amounted to 26.5 mmol·kg^-1. During the fermentation, a higher concentration of ammonia was noticed in groups D₂ and D₁ than in the control group. This phenomenon may be explained by a capacity of the preparation for absorbing NH₃.

According to Garcia et al [36] a level of ammonia in the rabbit caecum oscillates between 1.86–23.9 mmol·l^-1, yet, according to other authors, it may be even higher than the value mentioned. For example, according to Morisse et al [63], the ammonia concentration in the rabbit caecum grows (from 12.9 to 28.8 mmol·kg^-1 of the caecum chyme) with food rich in fibre. Gidenne [42] achieved the ammonia concentration in the rabbit caecum at a level of 23.6 mmol·l^-1 when he fed the rabbits with feed with 90% of the lucerne content. The high level of ammonia in the caecum chyme may be explained by a low ratio of digestible energy to digestible proteins in such food compared to the requirements of the growing rabbits. The value of this factor decreases with an increase in the participation of crude protein in feed, with a simultaneous increase of the VFA concentration [35,37,41]. When protein ingestion exceeds its requirements, the return of urea with blood to the caecum may increase leading to an increase of the ammonia concentration [30,31,33]. The concentration of ammonia in the rabbit caecum also depends on a type of fibre supplied with feed. In the experiment of Garcia et al [35], a decrease in the ammonia concentration in the rabbit caecum with an increase of the NDF content in lucerne given to the animals was observed.

In the research conducted by Marounek et al [56,57], the average ammonia concentration in the rabbit caecum was close to the one obtained in this research and amounted to 25.5 mmol·kg^-1 of the chyme. While examining the contents of the caecum in the rabbits aged 6 and 16 weeks, Bellier et al [5] found a decrease of the ammonia content with the age of an animal as opposed to the VFA content, which grew at the same time. Bennegadi-Laurent et al [6] obtained decrease by 20 % in the ammonia concentration in the rabbit caecum between the 28th day and 70th day of the rabbit life. However, Gidenne and Bellier [38], who used the caecal cannulation in their experiment, found the highest level of ammonia in the caecum chyme of the adult rabbits (approx. 3 kg) with an empty stomach, which amounted to 24.4 mmol·l^-1 on the average, whereas the same value was half as high (11.5 mmol·l^-1) in the case of the young seven-week-old rabbits (approx. 1 kg). The differences decreased after the morning meal and reversed 9 hours later, and the ammonia concentration decreased by 75% at the same time.

In the research conducted by Murray at al [65], which regarded an influence of bentonite on the parameters of the rumen fermentation in sheep, no significant differences in the ammonia content were found in the animals that were given this additive in the sheep’s feed (2.5 %) compared to the control group. However, the other authors observed a slight influence of the added bentonite (2 % of the feed) on an increase in the concentration of nitrogen contained in ammonia in the sheep rumen [46]. In the research conducted by Coffey et al [18], the addition of magnesium-potassium silicate to the heifers’ feed (2.5 and 10 %) caused an significant increase in the level of ammonia in the rumen.

Unlike VFA, the content of lactic acid is high from the duodenum to the ileum, whereas a lower concentration of this compound is found in the large intestine. In the Vernay’s research [82], the concentrations of lactate in the caecum during the phases of production of the soft faeces and hard faeces did not differ statistically and amounted to 3.8 and 3.6 mmol·l^-1 of the intestine chyme, respectively. A similar concentration of the above-mentioned acid in the caecum chyme was obtained by Piatoni et al [71], who found it to amount to 3.8 mmol·kg^-1 on the average in the 56th day of the rabbit life. In this research, the lactate concentration prior to the fermentation of the caecum chyme had a value close to the above-mentioned values and amounted to approx. 3.63 mmol·kg^-1. Under the influence of Humobentofet, the content of lactic acid grew. In the caecum, where the lactic acid is one of the precursors of the propionate [80], the increase of the level of both acids under the influence of the same factors seems to be justified.

Reaction of the caecum chyme determined in this research decreased in the course of fermentation in all groups, as well as under the influence of HBF compared to the control group. One could expect that the concentrations of VFA, lactic acid and NH₃ in the caecum have a decisive influence on the caecal pH, as they are the main sources of H⁺ and OH^¯. According to the data from UPM in Madrid [36], pH of the caecal chyme in rabbit shows a falling tendency when the concentration of VFA grows and the concentration NH₃ falls, yet these differences explain only 12% of the pH changeability in this intestine. Such a result suggests an impact of other factors on the caecum pH such as the physicochemical characteristics of the dry caecal contents, which is closely correlated with proportion of the particles smaller than 0.3 mm. The negative correlation between pH and the total concentration of VFA was found, yet no correlation between pH and the ammonia level was found in the rabbit caecum [6].

Reduction of pH was also received during the rumen fermentation in sheep under the influence of only bentonite [65], which would suggest a decisive impact of this ingredient of Humobentofet on the pH reaction of the chyme of the rabbit digestive tract. Such a conclusion seems to be confirmed by the already mentioned research carried out by Falcão-e-Cunha et al [27], in which an influence of fat on the value of pH of the chyme in the rabbit caecum was not noticed. Reduction of pH within such a scope as in this research, caused favourable modification of the caecum microflora and inhibition of multiplication of the pathogenic strains, which, as a result, contributes to the prevention of diarrhoea. Slight reduction of the reaction of the large intestine chyme and stabilization of the local microflora is also achieved through the addition of prebiotics to the animals’ feed, for example, oligosaccharides [61].

CONCLUSIONS

Summing up, it should be stated that the humic-mineral-fatty supplement did not cause any unfavourable changes in the bacterial processes during the in vitro fermentation in the chyme of the rabbit caecum. The observed changes concerning the determined fermentation parameters seem to be favourable and one can presume that they would positively influence the productivity of the species.

The most important effects of the influence of the mineral-fatty feed supplement on the in vitro fermentation in the chyme of the rabbit caecum were the following: the increase in the participation of propionate in the total concentration of VFA, the increase of fermentation efficiency, the rise of content of the glycogenic VFA compared to the non-glycogenic ones and the lowering of the pH value. The changes mentioned above prove the favourable impact of Humobentofet on the bacterial processes that take place in the examined sections of rabbit digestive tract, especially in the case of animals bred for consumer purposes.

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Accepted for print: 7.11.2007

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