The complex microbiota of the gastrointestinal tract (GIT) of animals plays an important role in the digestion of nutrients and protection against infections resulting from the presence of pathogens and environmental bacteria. It also functions as a barrier against harmful exogenous substances and ensures normal metabolic, immune and neurological functions in the host. The GIT is involved in numerous physiological processes, from nutrition to behavioural and stress responses [1
]. The unique digestive properties of rabbits and specialised microbial communities can help them to adapt to fibre-rich foods but often make them susceptible to metabolic diseases. For this reason, control and modulation of the microbiota of rabbits is an important aspect of breeding practice. Digestive problems can be controlled through the immunostimulatory activity of the microbiota and competitive exclusion, which is particularly important in young animals after weaning.
As in the case of all mammals, the introduction of new species to the GIT of rabbits is determined by ecological succession. Microorganisms, competing for nutrients, colonise an ecological niche and consume all food resources. These bacteria are able to inhibit the growth of competing bacteria by producing antimicrobial substances [2
]. A community of intestinal microbes is formed by random colonisation from the surrounding meta-community (the mother, bed, cage, air, etc.) and is highly variable between individuals up to the age of 49 days [3
]. By the age of 70 days, the composition of the caecal microbiota is highly homogeneous and shows a certain degree of stabilisation. In rabbits, a disturbance of this normal microbiota, known as dysbiosis, is widely considered to be the cause of enteritis with symptoms of diarrhoea, followed by dehydration and potentially death. The aetiopathogenesis of intestinal inflammation in rabbits is complex, and there are generally multiple factors. Enteric pathogens, such as Escherichia coli
, Clostridium spiroforme
, Lawsonia intracellularis
, Clostridium piliforme
spp., rotaviruses, coronaviruses, parvoviruses and astroviruses, are most often found in individuals with diarrhoea. However, there is still little information on the factors that initiate it, as infection with these microbes is not synonymous with disease. Most cases of enteritis in rabbits are caused by a combination of multiple environmental factors and infectious agents, including a low-fibre diet, an overall weakened state of health, stress associated with management and inadequate welfare, and age, as well as the presence of one or more potentially pathogenic microbes [2
During microbiological fermentation in favourable intestinal conditions, rabbits receive products that stimulate colonization by symbiotic microbes. In rabbits, this is referred to as a combined model of competition and cooperation of the gut microbiota. However, the balance of this ecosystem is fragile and can be destroyed during digestive disorders [5
]. An appropriate diet therefore plays a key role in prevention, which is why breeders often use special feed additives, such as probiotics, prebiotics or synbiotics, to improve gastrointestinal function. These additives, due to the fact of their antagonistic effect on pathogenic and opportunistic microbes, are the subject of growing interest among livestock breeders.
Improvement in the efficiency of digestion through optimisation of the composition of the microbiota directly improves nutrient digestibility and stimulates immune processes, increasing the profitability of production. This is one of the main reasons that breeders are searching for alternative solutions involving administration of probiotic microbes with feed in the form of monocultures or a mixture of different strains. The most commonly used probiotics in breeding practice include species of the genera Lactobacillus
. Work is also being conducted on recombined probiotics, which are among the most innovative biomedical applications of genetically modified organisms [6
Rabbits, like other farmed animals, are fed complete pelleted feed containing soybean meal (SBM) as the main source of protein. In some parts of the world, especially where soybean is not cultivated, recent years have seen a trend towards elimination of SBM from feed for most livestock animals. This is due to the desire to become independent of imported feed or to concerns about products containing genetically modified organisms [7
]. In the diet of rabbits, the possibility of replacing SBM with other high-protein plant-based feeds has been studied. The use of dried kernels of barley, wheat and maize, white lupin seeds [8
], peas [10
] or other plants of the Fabaceae family [13
] has been shown to have a beneficial effect on production efficiency. However, a large share of these ingredients in the diet results in health problems associated with the presence of anti-nutrients (e.g., tannins, antitrypsin factor, haemagglutinin, α-galactosides and alkaloids) or problems involving the balancing of the diet [15
Fermented protein components are currently a subject of great interest. Owing to their synergistic effect involving stabilisation of the gut microbiota and their valuable nutritional properties, they are becoming a sought-after bioproduct in the feed market. At the same time, efforts to limit the use of animal feed containing genetically modified organisms (GMOs) have prompted the search for an alternative source of easily digestible protein. A new material that meets these expectations may be fermented rapeseed meal (FRSM). Due to the microbial fermentation process, rapeseed meal (RSM), on the one hand, loses its anti-nutritional substances and, on the other hand, acquires probiotic properties. Moreover, it becomes a source of sulphur-containing amino acids, more easily digestible protein, digestive enzymes, and antioxidant compounds. In research in pigs, its inclusion in the diet has been shown to improve nutrient digestibility, resulting in improved growth performance. At the same time, by reducing unfavourable gut microbes and stimulating immune processes, FRSM has a prophylactic function and positively affects animal health [18
Bacteria of the genus Bacillus
, used in fermentation of plants, can perform a probiotic function, and the products of their metabolism can beneficially modulate immune system activity in animals [18
]. De-Yu Hung et al. [19
] demonstrated that Bacillus
bacteria have a positive effect by alleviating diarrhoea and reducing the number of gut pathogens. The studies cited show that FRSM can partially replace SBM in diets for monogastric animals and can be a valuable additive stimulating immune processes in the body and, thus, improve the condition of animals.
The available literature lacks studies on the possibility of using FRSM in the diet of rabbits. Moreover, there are few studies characterising the gut microbiota of rabbits, and these often focus only on individual segments of the GIT. Therefore, the present study was conducted to determine the effect of the use of varied amounts of FRSM in diets for rabbits on the immune status and microbiota of segments of the GIT, i.e., the duodenum, small intestine, caecum and colon.
An essential factor for maintaining a balance of the populations of the microbiota is a diet with an appropriate composition. Disturbances of their activity may result from the presence of anti-nutritional substances in feed components such as glucosinolates or tannins in RSM. During fermentation, anti-nutritional substances undergo reduction, leading to an increase in the level of lactic acid that exerts a beneficial effect by stimulating the immune system. FRSM, owing to its nutritional value and health-promoting properties, can beneficially affect production parameters, nutrient digestibility, the gut microbiota and metabolic processes [18
During digestion in rabbits, changes take place in physicochemical parameters, such as pH, redox potential, and metabolite concentrations, that directly affect the balance of microbial communities in the GIT [32
]. Michelland et al. [33
] demonstrated that the bacterial community of the caecum of rabbits can change and adapt to varying qualities and quantities of nutrients in the diet and can achieve a balance quickly following intake of new feed. This was confirmed in the present study in which the inclusion of FRSM in the diet of rabbits was not shown to affect most key groups of microorganisms taking part in feed digestion.
The microbial species variation in the GIT of rabbits is affected by numerous factors, from intestinal peristalsis and mixing of the intestinal contents determined by physiological structure, to periodic gastrointestinal strictures (as in the duodenum), to the presence of a number of gastrointestinal hormones and peptides, such as cholecystokinin, somatostatin, vasoactive intestinal peptide and “substance P” [34
]. Bowel transit time is highly variable and shorter than in other herbivores. Peristaltic contractions occur slowly, every 10–15 min, and do not change with the stages of the caecotrophic cycle. Food retention times are estimated at 10–20 min in the jejunum and 30–60 min in the ileum [35
]. The type and number of microorganisms in the GIT depends on the section of the GIT. In its first sections, i.e., the stomach and superior part of the duodenum, due to their acidic pH, the presence of bile, shorter transit time and limited mucus production, the number of microbes is smaller. These sections of the GIT are dominated by lactobacilli and enterococci. From the superior part of the duodenum to the jejunum and ileum, the number of bacteria steadily increases, until it reaches a value from 1011
CFU/g of faeces in the colon and caecum. The availability of oxygen and changes in pH in different sections of the GIT are conducive to the development of Bacteroides
spp., which carry out fermentation processes leading to the generation of volatile fatty acids. These, in turn, regulate intestinal pH, serve as an energy source for intestinal epithelial cells and promote gastrointestinal motility [36
A high level of high-quality fibre (NDSF) and protein in the diet of rabbits is important as well. Intake of rapidly fermenting fibre has a beneficial effect on the activity and concentration of volatile fatty acids in the caecum and improves the functioning of the intestinal mucosa and the structure of the microbiota. A diet with a well-balanced content of protein and essential amino acids, rich in arginine, reduces the population of Clostridium
spp. and Helicobacter
spp. bacteria in the intestines, thereby reducing mortality in the herd [37
]. The diet proposed in the present study, containing FRSM (8–12%), can be a valuable source of these substances that benefit rabbit health and can play a role in preventing imbalances in the gut microbiota. The probiotic and prebiotic substances contained in it are believed to stimulate the growth of Bifidobacterium
bacteria, which are beneficial for the host. Probiotics have been shown to have a positive effect on the gut microbiota of rabbits in research by Bónai et al. [38
]. The authors observed a decrease in the number of anaerobic Clostridium
spp. and E. coli
bacteria following administration of inulin. These results are consistent with those obtained in the present study. Rabbits from the groups receiving a diet with an increased proportion of FRSM (8% or 12%) had lower concentrations of anaerobic bacteria and E. coli
in the intestinal contents. Bacteria of the genera Bacillus
, used in fermentation of rapeseed, may perform an important probiotic role in the GIT of rabbits. The biological effects of their activity depend on the strains of microorganisms and their ability to maintain metabolic activity in the gut environment. Studies by Bónai et al. [39
] and Pascual et al. [40
] indicate that the addition of a probiotic in the form of Bacillus cereus
bacteria or Saccharomyces cerevisiae
yeast to the diet of rabbits has a beneficial effect on their health. The authors observed a decrease in the number of Clostridium
and E. coli
bacteria, which is in agreement with the results of our experiment. Research by Kimse et al. [41
], on the other hand, found that probiotic bacteria did not affect the structure or diversity of the bacterial population in the intestine of rabbits. In the present study as well, the probiotic microorganisms contained in the FRSM had no significant effect on the size of the major groups of microorganisms or their total number. Although the occurrence of Lactobacillus
strains in the rabbit microbiota is low, supplementation with them increases the number of cellulolytic bacteria and increases the number of ureolytic bacteria [42
]. In the present study, a significant increase in the number of lactic acid bacteria in the duodenum and colon was observed as the proportion of FRSM in the diet increased.
The gut microbiota is closely linked to the immune system (gut-associated lymphoid tissue—GALT) and in most mammals is primarily located in the small intestine and colon. Rabbits have special additional structures—the sacculus rotundus, located at the ileocaecal junction, and the vermiform appendix, at the end of the caecum. Together with the gut microbiota, lymphoid aggregates, such as Peyer’s patches and isolated cells dispersed in the lamina propria and the epithelium of the villi, play an important role in the acquisition of immunity and differentiation of antibodies [43
]. FRSM, rich in bioactive peptides, can stimulate the activity of the intestinal immune system (GALT). By stimulating the microbiota of the GIT and reducing the proportion of potentially pathogenic microbes, such as Clostridium
or E. coli
bacteria, it can counteract excessive immunisation in the intestinal mucosa and prevent inflammation. This is confirmed by Stappenbeck et al. [44
], who observed a decrease in GALT activity in mice that were not exposed to pathogens. The authors also showed weak immunisation of the immune system and a low level of antibodies in the blood serum. In rabbits, levels of antibodies are dependent on the gut microbiota, which influences the level and diversification of the repertoire of antibodies. A study by Severson et al. [45
] showed that the GALT system, despite continual immunisation by the commensal bacteria of the digestive tract, is able to acquire tolerance to Bacillus
antigens with no increase in the immune response. This is confirmed by the present study using RSM fermented with Bacillus
strains. This suggests that tolerance to the antigens of these microbes can be acquired, and the strength and direction of the effect of FRSM was established based on correlation analysis. The formation of mechanisms of tolerance also depends on the presence of probiotic microorganisms and the substances they produce.
FRSM, by supplying valuable bioactive substances, appears to be a good component in the diet of rabbits, enhancing immune system development and helping to prevent disturbances of the gut microbiota. The results of the present study indicate that the use of new biotechnological research solutions can lead to changes in agricultural practices. The use of fermented feed products in the diet can diversify protein sources and stimulate the beneficial microbiota of the GIT of animals. These measures are particularly important because they can help to reduce the use of antibiotics for therapeutic purposes through nutritional prevention and optimal development of the immune system.