Phytogenic and Probiotic Feed Additives for Broilers: Evidence for Growth Performance Links with Gut Performance Indices
It is becoming evident that zootechnical performance is directly linked with gut performance. The latter is determined by the continuous interaction between diet, gut microbiota, gut mucosa and the animal’s immune system. Certain bioactive feed additives elicit direct and indirect biological responses that are purported to aid in fine synchronizing key functions of the four interacting elements stated above and enhance performance. In this sense, research cases demonstrating links between growth performance, nutrient availability and modulation of key gut ecology and immune related indices upon phytogenic and probiotic administration in broiler chickens will be presented.
It is common understanding that beyond the requirement for adequate and balanced nutrition, animal performance can only be realized via an improved gut function and health. The term “gut performance” has thus been coined to precisely highlight the link between zootechnical performance and indices pertaining to optimal gut function and health.
Optimal gut function and health status
The critical question that remains to be answered is, how can an optimal gut function and health status be defined and captured? According to contemporary research, such an optimal status could be achieved only when certain dynamic and continuously interacting components of the gut ecosystem, such as dietary factors, gut microbiota, gut mucosa and immune response (Figure 1) are fine-tuned and reach an equilibrium that favors enhanced nutrient utilization, pathogen elimination, host detoxification and management of inflammation (Koutsos and Arias, 2006, Choct, 2009, Applegate et al., 2010).
Gut performance biomarkers
Each one of the major critical interacting components within the gut is a multi-element component that contributes to the high overall complexity of the gut ecosystem (Figure 1). For example, gut mucosa functionality is concomitant with an efficient gut barrier that in turn depends on gut morphology, integrity of epithelial cells, gut mucus layer, mucin composition, bacterial surveillance and the regulation of the inflammatory immune response. Therefore, in order to capture snapshots of the gut function and health status, there is clearly the need to identify key responses – biomarkers within the multi-elements underpinning gut performance that could be readily and reliably determined. Such biomarkers currently under investigation include: a) broiler digestive capacity, nutrient and energy utilization (e.g. nutrient digestibility, protein efficiency, energy efficiency and expression of key nutrient transporters), b) gut microbiota composition (e.g. luminal and mucosa-associated microbial species) and metabolic activities (e.g. volatile fatty acids, expression of microbial enzymes), c) epithelial histo-morphology and ultra structure, d) gut integrity (e.g. tight junction proteins), e) gut mucin composition, f) capacity to inhibit pathogenic colonization, antioxidant status and immune regulation (e.g. toll like receptors, cytokine gene expression, sIgA) at gut local and systemic level.
The gut performance biomarkers above could then be related to the corresponding growth performance responses and thus provide useful links between growth performance, nutrient availability and modulation of key gut ecology and immune related indices.
Challenges and opportunities for sustainable production
The worldwide demand for livestock products is projected to more than double by the year 2050, given the forecasted rise of human population to 9 billion. Poultry meat and poultry processed products are expected to have a strong increase in demand. The latter will occur as a result of competitive pricing, diversity of available products and the fact that poultry products are acceptable worldwide irrespective of religious or other cultural issues.
On the other hand, certain challenges such as rising production costs, environmental issues and climate change, animal welfare and the increasing worldwide pressure for removal of antibiotic growth promoters (AGP) need to be effectively tackled in order to maintain a sustainable production of poultry products. Likely, it is now possible to maintain and even optimize broiler growth performance, minimize economic losses and ensure the safety of broiler meat via proper combination(s) of certain intervention strategies, such as genetic selection of resistant animals, sanitation practices, elimination of pathogens from feed and water, vaccinations and applications of suitable feed and water additives.
Bioactive feed additives targeting gut performance
Both phytogenics and probiotics are powerful examples of bioactive feed additives (Windisch et al., 2008, Yang et al., 2009, Applegate et al., 2010, Hippenstiel et al., 2011), that when utilized as part of functional feeding strategies have the potential to beneficially modulate certain gut ecosystem elements through their properties to exert their effects at gut luminal, mucosal and systemic level.
Phytogenics in particular comprise parts of various aromatic plants and spices as well as fruits and/or their respective plant extract bioactive constituents (e.g. essential oils, oleoresins and flavonoids). Examples of applications of phytogenic feed additives (PFA) in broiler diets include oregano, thyme, rosemary, sage, anise, cinnamon, citrus and pepper plant parts or respective plant extracts (e.g. carvacrol, thymol, menthol, anethole and limonene) but also as blended combinations of multiple phytogenic compounds.
Probiotics on the other hand represent a functional nutritional approach, whereby maintenance of a healthy gastrointestinal environment and an improved intestinal function is pursued through the intake of adequate quantities of beneficial microorganisms. Examples of probiotic applications in poultry include microbial species belonging to Lactobacillus, Streptococcus, Bacillus, Bifidobacterium, Enterococcus, Pediococcus, Aspergillus, and Saccharomyces administered either as individual species/strains or as multi species/stain combinations.
A growing body of scientific literature supports the role of phytogenics and probiotics as alternatives to AGP in poultry and other livestock. It is becoming increasingly evident that among the most crucial factors that affect the efficacy of phytogenic and probiotic applications in broilers are primarily their composition (e.g. species/strains etc.) and administration levels. In addition, other factors such as bird growth phase, overall farm management (e.g. breeding intensity, HACCP measures and hygiene), environmental stressors (e.g. temperature, humidity, stocking density) and overall diet composition and type (e.g. raw materials, mash or pellet form) could affect phytogenic and/or probiotic efficacy.
In addition to the “macro-level” of growth performance determination, current research is focusing in the “micro-level” of gut performance biomarkers in order to generate new knowledge on the mechanisms underpinning the potential benefits evidenced for growth performance. The following sections of this paper will briefly overview six collaborative trials with phytogenic and probiotic applications in broilers that have been contacted in our laboratory with the focus to highlight links between broiler performance and some critical gut performance biomarkers studied.
Case studies of phytogenic applications
In this section, results from three experimental trial cases with phytogenic feed additives (PFA) will be summarized (Table 1). In experimental trial 1, a PFA based on a blend of oregano, anise and citrus essential oils with carvacrol, anethol and limonen being the main active ingredients and fructo-oligosaccharides acting as a carrier (Biomin® P.E.P., BIOMIN GmbH, Austria) was administered at 3 inclusion levels (i.e. 80, 125 and 250 mg/kg diet) in broiler diets. Experimental trial 1 included a non PFA supplemented group as negative control and a group that received AGP (i.e. avilamycin) as a positive control. Experimental diets were in a mash form and there was no coccidiostat included. In experimental trials 2 and 3, a PFA that contained components based on herbs, spices and essential oils characterized by menthol and anethole (Digestarom® Poultry, BIOMIN Phytogenics GmbH, Germany), was used at 2 inclusion levels (i.e. 100 and 200 mg/kg diet) in broiler diets. Trials 2 and 3 differed only in the cereal used (i.e. trial 2 maize was used instead of wheat in trial 3). Both trials 2 and 3 included a non PFA supplemented group as negative control and all diets had coccidiostat included. All trials used male Cobb-500 broilers and mash diets provided ad libitum and trial duration was 42 days.
In the first trial it was shown that overall FCR differed among treatments. Phytogenic inclusion level at 125 and 250 mg/kg diet improved FCR by up to 6.8% compared to the negative control group and did not differ from AGP (Mountzouris et al., 2011). Phytogenics improved overall body weight gain by up to 7.6% in maize diets (trial 2; Paraskeuas et al., manuscript in preparation). Depending on the inclusion level an improved overall FCR by up to 6.8% was also seen in wheat based diets (trial 3). In addition in the latter case, a reduced FI was evidenced (Paraskeuas et al., 2016).
Trials 1 and 3 despite using different phytogenic preparations have confirmed the general trend reported in scientific literature, whereby the improvements in FCR come as the result of reduced FI at a largely unchanged BWG (Brenes and Roura, 2010). The digestibility studies carried out within each one of the 3 trials showed that phytogenic inclusion level may affect positively the available dietary energy to the birds (Mountzouris et al., 2011, Paraskeuas et al., manuscript in preparation). Phytogenic inclusion level also affected key components of the gut microbiota in trial 1 and trial 3. Microbiological analysis by culture method techniques showed that although PFA inclusion did not affect cecal total anaerobes, coliforms and clostridia, yet it did affect Lactobacillus spp and Bifidobacterium spp levels by actually increasing their counts in a linear pattern with increasing PFA inclusion level (Mountzouris et al., 2011). In addition in trial 3, it was shown that PFA increased the levels of cecal total bacteria and members of the Clostridium coccoides subgroup determined by real time PCR (Paraskeuas et al., manuscript in preparation).
Moreover, PFA inclusion level did have a significant effect on small intestinal mucin composition of 14 d old birds. Mucin is the major mucus component. It is well known that intestinal mucus constitutes the first line of defense against luminal threats. Mucins are high-molecular-weight glycoproteins that are synthesized, stored and secreted by goblet cells of the enteric epithelium. They are characterized by a protein backbone and a high proportion of O-linked carbohydrates (50 to 80%). Five different monosaccharides are commonly found in mucins, namely N-acetylgalactosamine (GalNac), N-acetylglucosamine (GlcNac), galactose (Gal), fucose (Fuc) and sialic acids (NeuAc). Mannose is found in N-linked configuration and in smaller amounts in intestinal glycoproteins. In this study, we isolated mucin and determined its monomeric carbohydrate components by HPLC. Significant effects were seen in duodenum and ileum with increased mannose and galactose molar ratios compared to the negative control group (Tsirtsikos et al., 2012a). It is worth mentioning that human breast milk contains more than 130 oligosaccharides that consist from similar to mucin carbohydrate components. The resistance of breast fed infants to enteric infections as well as the establishment of a healthy gut microbiota is largely attributed to these oligosaccharides (Mountzouris et al., 2002). Therefore, the extent to which the changes shown above can mediate resistance to pathogenic challenges remain to be elucidated.
In the case of maize diets (trial 2) changes in gene expression of Muc2 and IgA in 42 d old ileal broiler mucosa were seen depending on the PFA inclusion level with the 100 mg/kg inclusion level having the higher relative expressions (Paraskeuas et al., manuscript in preparation). Interestingly, in trial 2 some early evidence for patterns of systemic anti-inflammatory effects were shown by the down regulation of IL18 and iNOS genes in spleen that however did not reach statistical significance at 5%. Nevertheless, an assessment of the changes above in conjunction with the significantly higher systemic total antioxidant (i.e. blood plasma) capacity conferred by PFA inclusion may provide evidence that PFA could contribute towards an overall better broiler health. In addition, studies in meat contacted in trial 1 confirmed the ability of PFA to increase the antioxidant capacity in meat (Table 1).
Case studies of probiotic applications
In this section, results from three experimental trial cases with probiotics will be summarized (Table 2). In experimental trial 1, a five-bacterial species probiotic that comprised probiotic bacteria isolated from the crop (Lactobacillus reuteri DSM 16350), jejunum (Enterococcus faecium DSM 16211), ileum (Bifidobacterium animalis DSM 16284) and ceca (Pediococcus acidilactici DSM 16210 and Lactobacillus salivarius DSM 16351) of healthy adult chickens (PoultryStar® me, BIOMIN GmbH, Austria) was administered at 3 inclusion levels (i.e. 108, 109 and 1010 CFU/kg diet) in broiler diets. Experimental trial 1 included a non probiotic supplemented group as negative control and a group that received AGP (i.e. avilamycin) as a positive control. Experimental diets were in a mash form and there was no coccidiostat included. In experimental trial 2, a three-bacterial species probiotic that consisted of Enterococcus faecium DSM21913, Bifidobacterium animalis DSM16284 and Lactobacillus salivarius DSM16351 isolated from the jejunum, ileum and caeca of healthy adult chickens (PoultryStar®, BIOMIN GmbH, Austria) was administered at 2 inclusion levels (i.e. 108 and 109 CFU/kg diet) in broiler diets. In a similar manner to trial 1, trial 2 included a negative and a positive control group, experimental diets were in a mash form and there was no coccidiostat included. In experimental trial 3, the inclusion of a probiotic similar to that of trial 1 was evaluated at 108 CFU/kg diet in broiler diets in combination or not with avilamycin used as an AGP. In addition, trial 3 tested also the thermally inactivated form of the probiotic also in combination or not with avilamycin used as an AGP. Experimental diets of trial 3 contained coccidiostat. All probiotic trials used Cobb-500 broilers, diets were in a mash form and were provided ad libitum during the 42 days of trial duration.
Probiotic administration in trial 1 resulted in significant improvements for overall body weight gain (i.e. up to 5.9%) overall FCR (i.e. up to 5.1%) and the European production efficiency factor (EPEF up to 9%). The responses above were dependent on the probiotic inclusion level. In particular, it was shown that under the experimental conditions and in the absence of a coccidiostat, probiotic inclusion level at 108 CFU/kg diet was the best for zootechnical performance (Mountzouris et al., 2010). Similarly, performance improvements were also seen in trial 2 (Mountzouris et al., 2015) and trial 3 (Palamidi et al., 2016).
In trials 1 and 2, whereby more than one probiotic inclusion level was assessed, probiotic supplementation resulted in more dietary energy becoming available for the birds in line with the AGP used as positive control (Mountzouris et al., 2010; Mountzouris et al., 2015). In both trials significance compared to the negative control group was reached at an inclusion level of 109 CFU/kg diet.
Microbiological analysis using culture based techniques revealed that the probiotic inclusion level modulated the levels of certain cecal bacteria and in particular coliforms, Lactobacillus and Bifidobacterium. In all the three cases above a probiotic inclusion level of 1010 CFU/kg diet gave the highest levels (Mountzouris et al., 2010). In a similar manner, in trial 2, using the more powerful fluorescence in situ hybridization (FISH) molecular technique, an elevation in the levels of cecal bifidobacteria with increasing probiotic inclusion level were also seen (Mountzouris et al., 2015). In addition to changes in the cecal microbiota composition, cecal microbiota metabolic activity was also affected by the probiotic inclusion level. In particular, changes in the pattern of volatile fatty acids (VFA) were seen with probiotics resulting in less acetate, less propionate and higher butyrate in the cecal digesta (Mountzouris et al., 2015). In trial 3, key components of the ileal and cecal microbiota were studied using real time PCR. In this trial the probiotic or its heat inactivated counterpart was administered at 108 CFU/kg diet. DNA was extracted from the relevant intestinal digesta and total bacteria, Bacteroides sp., Lactobacillus sp., Bifidobacterium sp., Escherichia coli, Clostridium perfringens subgroup (Clostridium Cluster I), C. coccoides subgroup (Clostridium cluster XIVa), C. leptum subgroup (Clostridium cluster IV) were detected and quantified. The results showed that at a probiotic inclusion level of 108 CFU/kg diet, the viable probiotic group had higher cecal Lactobacillus levels, compared to the non supplemented control, while the inactivated probiotic group had intermediate levels. Interestingly, this study also demonstrated that the AGP reduced the ileal lactobacilli and the cecal C. perfrigens (Palamidi and Mountzouris, manuscript in preparation).
An increase in mucus thickness in the duodenum was shown with probiotic inclusion level, while no effects were seen in the ileum and ceca. However, the cecal mucin glycosylation pattern was affected significantly, with probiotic inclusion level resulting in reduced GlcNAc and galactose and increased fucose (Tsirtsikos et al., 2012b). As in the case of phytogenics, the relevance of the changes in the mucin glycosylation pattern with regards to potential host resistance to pathogens remains to be elucidated.
In trial 3, whereby broilers were administered viable or heat inactivated probiotic changes in the MUC2 gene expression were seen in the ileal and cecal mucosa. In particular, the viable probiotic preparation resulted in lower expression compared to its heat inactivated counterpart (Palamidi and Mountzouris, 2016 manuscript in preparation). The effect was more pronounced in the ceca where proliferation and higher metabolic stimulation occurs, thus most likely implying that besides the actual cell components certain metabolites associated with active living cells are required to get the full effect on the particular response. Finally, from the latter experiment the gene expression of an array of immune related cytokines and factors were studied in broiler cecal tonsils (e.g. birds’ secondary lymphoid tissue). The analysis demonstrated an anti-inflammatory function of the AGP resulting in lower iFN-γ and iNOS, while it was also shown that probiotics (viable or heat inactivated) in combination or not with AGP reduced inducible nitric oxide synthase 2 (iNOS). The latter may imply an anti-inflammatory effect exerted by viable or inactive probiotics and/or AGP at the local cecal level (Palamidi et al., 2016).
Nutritional strategies utilizing phytogenics and probiotics represent powerful approaches to modulate the gut ecosystem in a manner that favors broiler growth performance and health. With the advent of our knowledge on the exact mechanisms and modes of their action, phytogenics and probiotics hold much promise for a sustainable broiler meat production and the overall safety of the food chain.