GASTRIC MICROBIAL FERMENTATION IN RABBIT UNDER INFLUENCE OF DIETARY SUPPLEMENT – HUMOBENTOFET 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 analyse the selected parameters of in vitro fermentation in the rabbit's stomach and to determine the influence of Humobentofet, a humic-mineral-fatty supplement, on them.  The research was conducted on 54 rabbits of the Belgian giant breed, aged 6 months.  The samples of gastric 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.  The samples to be analysed were taken from bottles before the fermentation (0. h) and in the course of fermentation, i.e. after 4^th, 6^th, and 24^th 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.  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 gastric content is manifested 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, stomach fermentation, humic-mineral-fatty supplement.

INTRODUCTION

Caecotrophy and stomach fermentation in rabbit
Microbial fermentation of ingesta takes place mainly in herbivores and omnivores, but also in carnivores. Fermentation chamber can be localized cranial to the acid-secreting part of the stomach (forestomach fermenters: ruminants, camels, hippopotamuses, and some monkeys and marsupials) or the caecum and colon serve as chambers for a microbial action (hindgut fermenters: rodents, lagomorphs, horses, elephants, and all omnivores and carnivores) [8].

In forestomach fermenters, vitamins are synthesized and protein from microbial cells is digested and absorbed from the small intestine, but in hindgut fermenters there is a problem with digestion and absorption of amino acids and vitamins absorption in the hindgut. For making most of the microflora and the products of its activity, some mammals, like rodents, reingest their own faeces (coprophagy).  Lagomorphs and also some rodents (beaver, ground squirrel) practice more efficient faecal reingestion called caecotrophy. Therefore rabbit produces 2 types of faeces: soft and hard. Hard feces are constituted from poorly digestible or less nutritious ingesta particles whereas soft faeces are directly of caecal origin. Caecotrophy is the consumption of the soft faeces and, consequently, caecal contents [8,11,13,45].

Caecotrophy in rabbits begins at 3 weeks of age, when rabbits begin to consume solid food, then the soft faeces production increases and achieves the peak on 63^rd – 77^th day of age (25 g of dry matter/day) [12]. Soft faeces are excreted according to a daily rhythm, which is opposite to that of food intake and hard faces excretion. Caecotrophy occurs mainly during the light period, whereas feed intake and hard feces excretion occur during darkness [5,6,12,19].

Differentiation between soft and hard feces begins during the transit of digesta through the caecum and proximal colon. During hard feces excretion, water-soluble substances and fine particles (smaller than 300 µm according to Gidenne)[22], including microorganisms, tend to accumulate at the circumference. They are taken back to the caecum by antiperistaltic movements and retrograde flow. The larger fibre particles, being of lower density, tend to accumulate in the lumen and pass to the distal part of the colon. Here hard feces are formed. In contrast, the motility of both the caecal base and proximal colon decreases during the formation of soft faeces (cecotrophes). That is why soft faeces are formed mainly from the caecal content (only water absorption and some mineral components changes take place in colon) and are lower in fibre and higher in nutritious substances than hard feces [21,22].

The water, electrolyte and volatile fatty acids content changes daily in hindgut according to excretion pattern. During soft faeces production, the water, Na⁺ , Cl^- and VFA are absorbed, whereas K⁺  is secreted (like in other mammals). In the hard feces production, absorption of the water, Na, K, Cl and LKT increase and only low nutritive value material is excreted in this way there [44].

Soft faeces contain more protein, minerals and vitamins than hard faeces. Protein of soft faeces is high in essential amino acids such as lysine, sulphur amino acids or threonine. Cecotrophes consist of small pellets of 5-mm diameter, which rabbit can recognize. They are taken directly from the anus, swallowed without mastication, and stored in the fundus of the stomach for 3-6 hours. The faeces contain bacteria, which here continue their proliferation and fermentation of starch.

After getting into the stomach, soft faeces do not mix with other food as they are placed separately at the stomach fundus and are protected against digestion with a mucous envelope, which consists of vitamin B₁₂ complex with mucoproteides. The bacteria that are included in the cecotrophes here continue their proliferation and fermentation of starch producing milk acid as a final product.

The cecotrophes a sufficient buffering ability to maintain the pH at a level of approx. 6.0, despite the stomach environment is characterized by high acidity. The bacteria that occur inside the soft faeces secrete amylase into the gastric lumen, which adapts to the acid environment and achieves 88% of its activity at the pH 5.0, whereas amylase from saliva achieves only 30% of its activity in such an environment [13,25]. The pH of the gastric contents is the main factor limiting the activity of the bacterial amylase, which fades away entirely at the pH below 3.2. Although, according to the literature of the subject, the pH in the rabbit's stomach amounts to approx. 2, the buffering ability of food, soft faeces and saliva prevents direct acidifying of the contents [9]. During the fermentation of soft faeces in the stomach, phosphates are released, which cause an increase in the pH of the gastric contents up to the values at which amylase, especially the one that comes from the soft faeces microrganisms, may maintain considerable activity [9,25,46].

The bacteria that live in those sections of the digestive tract that precede the caecum were detected already on around the 17^th day of the rabbit's life [12,24]. A relatively high level of pectinolytic activity was also observed  in the stomach [32].

The fermentation which occurs at the fundus of the stomach is not a typical phenomenon for the whole Family Leporidae. In hares (Lepus), amorphous soft faeces without envelope are observed, which, after swallowing, are not recognizable in the gastric contents [26].

To sum up, cecotrophy guarantees a maximum use of feed both in the case of a high-fibre (low-energy) diet and a (high-fibre) diet with less fibre [40]. It can compensate for a low-quality protein or a low-vitamin diet in the traditional breeding as well as it is necessary to provide extra B vitamins, minerals and limiting amino acids in the intensive breeding [12,13].

The humic-mineral-fatty preparation
Feed supplements which enrich the nutritive value of food are recommended for animal feeding both in intensive and domestic breeding. The research on the use of natural materials and organic and mineral products in the animal feeding has been undertaken more and more often recently. It improves breeding parameters and does not have an adverse effect on stability of microflora in the digestive tract [37,49].

Humobentofet (HBF) is one of such preparations which are vegetable oil mixture on humic-mineral carrier. The main ingredients of Humobentofet are the humic compounds (humodetrynit) and fatty acids based on bentonite: oleic, linoleic, linolenic, palmitic and other. The addition of the humic-mineral-fatty supplement as well as other additives containing humic compounds has proved their favourable effects on feeding of fattening pigs, sows, cattle, sheep, and poultry [14,15,16,27,28,29,33,39,41,42]. The 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. Moreover HBF 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 beneficial effect on animal health and productivity causes being the application of Humobentofet also interesting for the rabbit feeding. The mineral composition of HBF includes 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 [15]. Moreover, a favourable effect of the unsaturated fatty acids on the rabbit's organism was found [17,18].

The earlier research concerning the effect of Humobentofet addition on rabbit caecal parameters [36]. There is the continuation of the study and the present research has aimed at determination of an impact of HBF on the in vitro fermentation parameters in the rabbit's stomach.

MATERIAL AND RESEARCH 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 [2]. The animals received water and feed ad libitum.

The contents of stomachs were taken directly after the animals' slaughter and then samples intended for examination were prepared, which included, each, 10 g of the digestive contents and 90 ml of solution containing the properly selected buffer compounds [1]. For the gastric contents, the pH of the buffer solution was lowered to a value of 4.7–5.0. An average value of pH in the rabbit's stomach amounts to approx. 2, yet after feeding, a buffering ability of food, soft faeces, and saliva prevents from such strong acidifying of the contents. After providing a large amount of food, the pH at a level from 4–4.5 to 5 was found in some areas of the stomach [9]. Acidifying of the contents below pH 4 is bactericidal to microorganisms of the rabbit's digestive [34], thus it makes the course of the fermentation process impossible.

Subsequently Humobentofet was added to the samples. The humic-mineral-fatty preparation was manufactured by the company PHW "Tronina" and is registered in MRiGŻ as a feed additive (ref. No. RRpp-4121-C-350/97). An energy value of Humobentofet amounts to 8MJ (1912 kcal)·kg^-1 [15].

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. The samples were flashed with nitrogen prior to the fermentation in order to achieve the anaerobic conditions. 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^th, 6^th and 24^th 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. 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.

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 chromatograms were registered by means of an Acord computer system.

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 and modified by Baran and Zitňan [3]:

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 [38] and modified also by Baran and Zitňan [3]:

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 [38]:

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.

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 and p≤0.01. 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

Unlike in the caecal contents, the pH-value of the fermented gastric contents did not change significantly under an influence of time or Humobentofet added (Table 1).

The concentration of the all studied volatile fatty acids in the rabbit gastric contents prior to the commencement of the in vitro fermentation reached a level of  30.21 mmol·kg^-1 (an average value of the groups K, D₁ and D₂ at 0. h of the fermentation; Table 1). The dynamics with which the value was changing in the course of the fermentation was different for the particular experimental groups and, at the same time, a significant impact of time on the course of the process was noticed (p≤0.01). In addition, significant differences under the influence of the time – added Humobentofet interaction were observed (p≤0.05). In the control group (K), without HBF supplemented, the level of volatile fatty acids was constantly growing and it reached 62.97 mmol·kg^-1 at 24 hours of the fermentation. In the groups with 10 % and 15 % of  HBF added (D₁ and D₂), the total concentration of the acids was also growing, yet until 6^th hour of the fermentation (thus within time in which the process proceeds in the physiological conditions) the growth was faster compared to the control group, whereas at 24 hours the lowest value of volatile fatty acids could be already observed in group D₂ (41.89) compared to the control group (62.97) and group D₁ (59.58 mmol·kg^-1).

Concentration of the acetic acid, which occurs in the highest amount in the contents of the rabbit's digestive tract, was growing in the course of the gastric contents fermentation (p≤0.01; Table 1). The significant differences in the level of C₂ were also noticed as a result of the time – added Humobentofet interaction (p≤0.01).

Additionally, significant differences in the percentage of C₂ under an influence of time and the added Humobentofet as well as the interaction of these two factors (p≤0.01) were observed (Fig. 1). After 24 hours of the fermentation, the greatest increase in the fraction of acetate in the total acetic, propionic and butyric acids was observed in the control group (i.e from 56.83% at 0. h to 77.5% at 24^th h), whereas, in groups D₁ i D_2, small differences in the percentage of this acid were observed during the fermentation (i.e. from 57.95 to 63.53 % and from 58.45 to 58.95%, respectively). Thus, an impact of HBF on lowering of the percentage of C₂ compared to the control group is evident.

Among the volatile fatty acids, the greatest changes in the fermented contents regarded the content of the propionic acid. A significant increase in the concentration of the acid was noticed both under the influence of time (p≤0.05) and the added Humobentofet (p≤0.01; Table 1). In the course of the fermentation, the level of C₃ increased considerably faster in the groups supplemented with HBF than in the control group, and it reached the highest value in group D₂.

Simultaneously with an increase of the propionate concentration under the impact of the HBF supplement, its percentage in the total three most important volatile fatty acids i.e. acetic, propionic and butyric acids also grew compared to the control group. In the course of the fermentation, both a significant influence of time (p≤0.05) and the feed supplement was noticed (p≤0.01), as well as an impact of their mutual interaction (p≤0.01) on the percentage of C₃ (Fig. 2).

A significant influence of the fermentation time on the concentration of butyric acid in the gastric contents was observed as well as an impact of the time – Humobentofet interaction on an increase in the level of this acid at the last stage of fermentation (p≤0.05) (Table 1). Analyzing the content of C₄ molar concentration in the total three most important volatile fatty acids, a significant influence of the fermentation time and the time – Humobentofet interaction on this value was also found (p≤0.01) (Fig. 3). During the first six hours, no significant differences were noticed, yet between 6^th and 24^th hours a drop in the content of butyrate was observed, fist of all, in the control group (from 28.98 to 15.01%) and in group D₁ (from 30.48 to 22.51%) as well, whereas the difference in group D₂ was small (30.47% at 6^th hours and 28.22% at 24^th hours).

Summing up of the percentage of molar concentrations of the acetic, propionic and butyric acids in their total, it can be stated that an increase in the percentage of acetate in the gastric contents was observed in the control group during the fermentation, which was lowered through the addition of Humobentofet. At the same time, the supplement increased the percentage of the two remaining acids (C₃ and C₄), which, during the fermentation of the gastric contents without HBF, showed a falling tendency.

In addition, the percentage of the above-mentioned acids is slightly different in the gastric contents from the caecal contents [36]. When the curves are compared which show the content of the above-mentioned acids in the control group during the 24-hour fermentation, an increase in the content of acetate in the gastric contents draws our attention at the cost of a decrease in the content of propionate and butyrate, whereas, in the caecal contents, the content of acetate was falling and the content of propionate was simultaneously growing.

A value of the propionate – butyrate molar concentrations ratio in the gastric contents did not show any significant differences under the influence of HBF, yet it was changing under the influence of the fermentation time  (p≤0.05) (Table 3). Prior to the fermentation, the ratio amounted to 0.44 on the average, yet it grew to 0.56 after the 24-hour fermentation.

The content of acids, which occurred in a slight amount in the chyme (iC₄, iC₅, C₅), was analyzed globally as 'min VFA' (Table 2). The total concentration of the acids significantly grew in the course of the gastric contents fermentation (from 0.43 mmol·kg^-1 at 0. hours to 6.3 at 24^th hours). In addition, at 4^th and 6^th hours  of the fermentation, the addition of Humobentofet influenced an increase in the level of the acids (especially in group D₁; p≤0.01).

The statistical analysis was not carried out for the concentrations of isobutyric acid and isovaleric acid individually as these acids were not found in most of the samples taken from the gastric contents of the rabbit. Taking into account a dynamics of changes to the course of the gastric contents fermentation for the valeric acid, it should be stated that the changes are similar with the ones in the case of min VFA, thus both time and the feed supplement influence an increase in a level of this acid (p≤0.01), however the influence of the supplement is limited to the 4^th and 6^th hour of the fermentation (Table 2).

Analyzing the remaining products of microbial activity, a significant influence of time (p≤0.05) but not the HBF supplement on the concentration of ammonia in the gastric contents was noticed (Table 2). Unlike the ammonia, an analysis of the lactic acid concentration showed that the influence of the HBF supplement proved to be significant (p≤0.01; Fig. 4), however, no impact of the fermentation time on the content of lactic acid was found. The lowest concentrations were observed in group D₂ (19.42 – 25.0 mmol·kg^-1l), whereas the highest ones – in the control group (26.24 – 42.74 mmol·kg^-1).

The fermentation efficiency FE, calculated on the basis the VFA concentrations determined in the gastric contents, was decreasing in the course of the fermentation (Fig. 5), however, the influence of the HBF supplement was definitely increasing it compared to the control group (Fig. 6, Table 3) (p≤0.01). Prior to the fermentation, the value of FE amounted to 74.2% in group K, 73.92% in D₁ and 74.01% in D₂, at 4. hours, respectively, 73.11, 75.05 and 74.44%, at 6. hours 73.02, 74.23 and 73.38%, at 24. hours: 69.31, 73.05 and 73.85%.

Like in the case of FE, a decrease in the value of E₁ (the energy stored in the VFA) was also observed in group K (p≤0.01) in the course of fermentation, whereas the HBF supplement clearly increased the value in groups D₁ and D₂ compared to the control group (the influence of the supplement  and the time – supplement interaction was significant for p≤0.01) (Table 3). The highest values were observed in group D₂ at 4^th and 24^th hours of the fermentation.

The value of E₂ which expresses the energy lost in the methane was approximately inversely proportional to E₁, thus it was increasing for the gastric contents in the course of the fermentation in the control group (Table 3), yet the Humobentofet addition was significantly decreasing the value of E₂ in groups D₁ and D₂ compared to the control group (p≤0.01). The lowest values of E₂ (the lowest loss of energy in methane) were noted for group D₂ at 4^th and 24^th hours of the fermentation.

The E₁/E₂ ratio, which informs about the yield of processes that occur in the chyme, increased under the influence of the Humobentofet added (p≤0.01; Fig. 7). A significant influence of the HBF supplement – fermentation time interaction was also noticed (p≤0.01) (Table 3). Prior to the fermentation, the E₁/E₂ ratio oscillated at a similar level in all groups and amounted to approx. 4.5.

Taking into account the influence of time on the fermentation efficiency in all group examined, the highest values for both FE and the E₁/E₂ ratio were noticed at 4^th hours of the gastric contents fermentation (Fig. 5 and 8).

In the case of NGGR which expresses the ratio of the non-glycogenic VFA/glycogenic VFA produced as a result of the fermentation, the significant influence of the HBF supplement (Fig. 9) and the HBF supplement – fermentation time interaction on the value of the above-mentioned parameter was observed in the gastric contents (p≤0.01). Prior to the fermentation, the value of NGGR was similar in all groups (8.35–8.79), yet, at 4^th hours, it grew to 10.05 in the control group, whereas it fell to 6.99 in groups D₁ and D₂ (Table 3). At 6^th and 24^th hours, the further increase in the value of the above-mentioned parameter was observed in group K (11.47 and 13.85, respectively), whereas in groups D₁ and D₂ it was subject to small alternations.

DISCUSSION

In the research on the products of the bacterial activity in the rabbit digestive tract that has been carried out so far, first of all, the large intestine was taken into account and there is little information on the content of these compounds in those sections of the digestive tract which precede the caecum. Yet, what is known is that, thanks to the presence of microorganisms in feeds, the fermentation of structural sugars takes place in the cardial section of a one-chamber stomach in pigs and horses, which leads to the formation of volatile fatty acids whose concentration may reach a value of 50 mmol·kg^-1 in a pig's stomach [4]. An influence of feed on the VFA profile was also observed in the pig's stomach, thus the feed in which beet pulp was used as a source of fibre caused a substantial increase in the production of acetic acid, but, at the same time, it did not influence a level of the remaining acids [47].

In this study, a lower level of the VFA was noted as well as slightly different proportions between particular acids in the gastric contents than in the caecal contents [36]. Prior to the fermentation (0. h), the content of VFA in the gastric contents amounted to 30.21 mmol·kg^-1, and the ratio of molar concentrations of the acids i.e. acetic, propionic, and butyric acids reached a value of: 58 : 13 : 29. The content of butyrate and propionate in the total concentration of C₂, C₃ and C₄ was greater here, whereas the content of acetate was lower than in the caecal contents. A value of the C₃/C₄ ratio was also slightly lower than in the caecal contents, which amounted to 0.44. The lower total concentration of the VFA in the rabbit gastric contents was obtained by Vernay [45], who found it at the fundus of the stomach at a level of 19.2 mmol·l^-1 at the stage of hard faeces production. The ratio of concentrations C₂ : C₃ : C₄ amounted to 66 : 14 : 20 and, like in this study, a lower content of acetate and a higher content of propionate were also observed here than in the caecal contents, where the proportions amounted to: 73 : 9 : 18, respectively.

Unlike in the caecal contents, where the addition of Humobentofet caused significant lowering of the pH-value during the fermentation [36], the pH-value of the gastric contents, studied in this research, did not significantly change during the fermentation under the influence of the supplement applied. Thus, the pH-value of the gastric contents seems to be more stable than the pH-value of the caecal contents, which is proved by the data coming from the literature. According to Mikkelsen at al [35], during the in vitro fermentation in the digestive track of piglets, the addition of oligosaccharides, which significantly lowered the pH-value during incubation of the contents with the colon and caecal bacteria, did not influence any changes to the pH-value of the contents with the gastric bacteria.

Among the VFA, the acetic acid occurs in the highest concentration in the digestive tract in most of animals. In ruminants as well as in pigs, horses, elephants, hippopotamuses, domestic birds, and in most of the rodents, including rats, coypus, and guinea pigs, the VFA profile is, additionally, characterized by predominance of the propionic acid over the butyric acid  [10,48]. However, a higher concentration of the butyrate than the propionate is a typical characteristic of not only the digestive tract of leporids but also mice, coypus or sea mammals [10,30,31].

During the in vitro fermentation, which occurs in closed systems, accumulation of the bacterial activity products takes place in the fermented digestive contents [1,43]. In this research, an increase in the molar concentrations of most of the studied volatile fatty acids was observed during the in vitro fermentation, yet their proportions were changing as well. In order to enable comparison of the above-mentioned observation with the data in the literature of the subject, the percentage of the concentrations of the three most important acids was compared to their total concentration in the digestive contents [7,20,23,40].

Comparing of the percentage of acetate in the total concentration of VFA in the gastric contents to the percentage of this acid in the ceacal contents, it should be stated that the percentage was growing in the gastric contents during the fermentation, whereas it was falling in the caecal contents [36]. The above-mentioned changes regard both the control groups and the average values received for groups: K, D₁, and D_2. The percentage of the propionic acid and butyric acid oscillated at the different levels in both sections of the digestive tract studied. The percentage of the above-mentioned acids was decreasing in the gastric contents during the fermentation, whereas, in the caecal contents, the percentage of propionate was growing and the percentage of butyrate did not show any significant changes. In both cases, the addition of Humobentofet lowered the percentage of acetate and increased the percentage of propionate.

Lactic acid was another determined product of the bacterial fermentation. This acid, which was found in blood, comes, first of all, from the gastric fermentation and, starting from the stomach, it joints the portal circulation, whereas the VFA are predominantly not absorbed before the large intestine [45,46]. Production of the lactic acid in the stomach takes place in the soft faeces, which was swallowed earlier, surrounded by mucous membrane, which enables it to remain untouched for at least 6 hours from swallowing. Unlike the ceacum, where most of the lactic acid is transformed, thanks to the bacterial processes, into VFA, which are considered to be the most important products of the fermentation in the large intestine, the lactic acid is the most important fermentation product in the stomach. In this research, the concentration of butyric acid was decreasing during the fermentation of the gastric contents with the addition of 15% of HBF.

In this research, the content of ammonia was considerably lower in the gastric contents before the fermentation than in the ceacal contents. This phenomenon may be explained by the prevailing amino-acid deamination processes in the ceacum, where part of these compounds may come from microorganism bodies and another part gets only here available to enzymes as a result of the bacterial degradation of cell walls  [43]. In this research, no significant differences in the ammonia concentration were observed during the gastric fermentation under the influence of the mineral-fatty supplement.

In this research, the analysis of the fermentation parameters calculated on the basis of the VFA concentrations showed that, like during the intestine contents fermentation, the fermentation efficiency in the samples of gastric contents increased thanks to the alternation of the fermentation profile under the influence of the mineral-fatty supplement as well as the ratio of the non-glycogenic VFA : glycogenic VFA (NGGR) got lower. Additionally, in the control group, the fermentation efficiency got lower and the utilization of VFA got worse during the fermentation in the rabbit's gastric contents, which was not observed during the fermentation in the rabbit's ceacal contents [36]. The reason for this phenomenon is probably microflora, which occurs in a greater number in the caecum and ferments nutrients.

CONCLUSIONS

To sum up the results of this research, one should state that changes to the proportions of the VFA during the in vitro fermentation of the rabbit's gastric contents show a different direction compared to the ones in the ceacal contents. The differences regard, first of all, the acetate whose percentage increases during the gastric fermentation whereas, in the caecal contents, it showed a downward tendency. In the case of propionate, the situation is entirely different. Its content decreases during the gastric fermentation, whereas it grew during the ceacal fermentation. The yield of the in vitro fermentation in the rabbit's gastric contents decreases during the fermentation and the utilization of VFA gets worse what was not observed during the fermentation in the ceacal contents.

Under the influence of the humic-mineral-fatty feed supplement, the percentage of propionate increases in the course of the fermentation whereas the percentage of acetate decreases. The content of butyrate significantly increases only between 6^th and 24^th hours of the fermentation. Thanks to the alternation of the fermentation profile under the influence of the mineral-fatty supplement, the yield of the fermentation in the gastric contents increases and the ratio of the non-glycogenic VFA/glycogenicVFA decreases.

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Accepted for print: 15.10.2008

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