The first week after weaning is the most the critical phase of weaning, when the diet changes from highly digestible (breast milk) to a more complex digestible solid food. This change directly affects the physiology of the piglets' gastrointestinal tract that is not fully adapted 15, causing intestinal and immune system dysfunction and result in less health, growth, and feed intake 16.The use of functional oils resulted in greater average daily weight gain (DWG) and feed conversion (FCR) in piglets during the first and second periods, which also resulted in heavier pigs at the end of the experiment.
Although it is not clear how the supplementation of functional oils improved pig growth, several mechanisms have been proposed, including antimicrobial activity, such as reducing pathogenic stress or increasing the abundance of beneficial microorganisms in the intestine, such as Lactobacillus spp1; protecting intestinal villi and regulating enzyme activity 17; also, modulating the intestinal microbiota and increasing the absorption of nutrients 18
Previous studies have shown that the supplementation of a blend of essentials oils (cinnamaldehyde and thymol) in the diet of weaned piglets positively influenced characteristics of zootechnical interest, such as higher DWG and lower FCR (P < 0.05)19,20,similarly to the results of the present study. Evaluating the supplementation of L. acidophilus in weaned piglets, 21 observed an improvement in DWG and FCR (P < 0.05) compared to the control group, and the same result was observed in the group receiving probiotics in the present study.
Probiotics act by modulating the microbiota, mainly by adhesion and competitive exclusion of pathogens at binding sites in the intestinal epithelium6. The blend of functional oils used in this study acts by modulating the immune system and the intestinal microbiota with antimicrobial action, mainly against gram-positive bacteria 11,13. Both additives provided better performance and modulation of the microbiota in the face of the weaning challenge due to different mechanisms of action.
In this study, there was no difference in microbial diversity between the additives, estimated by the Chao, Shannon and Simpson indices. Similar results were found by 22 and 23, who evaluated the supplementation of essential oils and probiotics in weaned piglets, respectively. Although without a statistical difference, there was a tendency to increased diversity for the group supplemented with functional oils. The increase in microbial richness and diversity can be seen as a predictor of the stability of the microbial ecosystem 24.
The phyla Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria, in decreasing order concerning relative abundance, are predominant in swine gastrointestinal tracts6. These results are in line with the findings in this study. The relative abundance of Firmicutes increased, while the relative abundance of Bacteroidetes decreased after supplementation with both additives. It has been shown that heavier pigs tend to have a higher Firmicutes vs. Bacteroidetes ratio than lighter animals 25, similar to what was observed in this study.
Functional oils modify the composition of intestinal microbiota, increasing the relative abundance of Firmicutes in the intestine, as demonstrated in broilers in vivo studies by 26 and in pigs by 22. It is necessary to highlight that the increase in the Firmicutes. Bacteroidetes ratio is a natural trend found in the healthy intestinal microbiota of matured piglets. However, in dysbiotic situations, such as those caused by weaning, it can result in a decrease in Firmicutes and an increase in Bacteroidetes. These changes provide a favorable environment for the proliferation of some pathogenic genera of this second phylum and, consequently, a reduction in the feeding efficiency of the animals 27.
Also, a large reduction in Bacteroidetes can cause damage to the host. Bacteroidetes, despite encompassing some pathogenic species, are known to have a large number of genes that encode active carbohydrate enzymes and can readily switch between different energy sources, in addition to being an important source of propionate 28,29. Additionally, Firmicutes has members nutritionally more specialized in the degradation of complex substrates, such as plant cell walls, starch particles and mucin 30. Therefore, a stable relationship between Firmicutes and Bacteroidetes can result in better utilization of the diet by animals. In the present study, both additives were effective in maintaining the Firmicutes. Bacteroidetes ratio, a fact that may have contributed to the better performance of the animals in both supplemented groups.
Tenericutes also seem to be involved in improving the use of nutrients by the host. In a study with piglets, 31 found a positive correlation of this phylum with a better apparent digestibility of crude fiber. However, data on the relationship of this phylum with animal performance are still scarce. Proteobacteria are known to harbor numerous opportunistic pathogens in animals and humans, including Escherichia coli, Escherichia Shigella, Salmonella, Brucella, Rickettsiaos spp. Thus, it is associated with several intestinal disorders and infectious diseases 32,33. In this study, the use of functional oils increased Tenericutes and inhibited Proteobacteria.
At the family level, supplementation with both additives kept the relative abundance of Lachnospiraceae stable, increased the Ruminococcaceae and Prevotelaceae abundances, and reduced Lactobacillaceae. These four families are known to be part of a group fundamental to the microbial activity in the piglets' intestines 34.
Ruminococcaceae, for example, are associated with fiber degradation and higher concentrations of butyrate in piglets35. Butyrate contributes to a better absorption of nutrients stimulating the growth of intestinal mucosa cells, improving the retention of calcium and phosphorus in the diet, mitigating the challenge of weaning 36 and inducing secretion of mucin, a glycoprotein, which forms a protective layer in enterocytes 37. The genus Faecalibacterium, from Ruminococcaceae, has been negatively associated with feed efficiency in pigs. In the present study, this genus was present in a higher percentage in the probiotics group when compared to the other treatments.
Prevotella and Lachnospiraceae are positively correlated with gene functions associated with the metabolism of amino acids, energy, cofactors and vitamins, indispensable to the host 38. Prevotella has also been positively associated with higher luminous IgA concentrations and body weight in weaned piglets, showing its importance to the health of piglets 38 .
Lactobacillus is prevalent in the fecal microbiota of piglets in early life and tends to decrease during the weaning transition 39. Several species of Lactobacillus are associated with beneficial characteristics for the host. It has been shown that the swine microbial population differs between more efficient and less efficient animals. More efficient animals have a higher number of Lactobacillus spp. than less efficient animals 40.
Interestingly, the opposite behavior was observed in the present study, where the blend and probiotics groups showed a less relative abundance of this genus (~ 3%), but better performance, while the control group, greater relative abundance (~ 9%) and less performance. The authors hypothesized that the higher concentration of Lactobacillus in the control group may be associated with the activation of the immune system of animals in this group in the face of a greater challenge (as evidenced by the higher rate of diarrheal incidence and E.coli count in feces of these animals). Many species of Lactobacilli act in the innate and acquired system stimulating immune cells to release pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ) and interleukin-12 (IL-12) 41. It is possible to conclude that the lower concentration of Lactobacillus in the functional oils and probiotics groups did not result in losses to the animals' performance.
The groups supplemented with Functional Oils and Probiotics showed a greater relative abundance of the Clostridiaceae family compared to control. This family is known to have different species, including C. pectinovor one, Clostridium butyricum, Clostridium perfringens. Clostridium butyricum, for example, acts in the production of short-chain fatty acids and has been studied as a probiotic in other animal species, such as broilers, where resulted in the improvement of the the function of the intestinal barrier and the inhibition of pathogens 42.
Curiously, the supplementation with probiotics (Lactobacillus spp., Bifidobacterium and Saccharomyces cerevisiae) did not result in an increase of these genera, other than for Bifidobacterium, in the fecal microbiota of piglets. Two factors may explain this result. On one hand, the technology and conditions involved in the preservation of these probiotics, which can negatively influence the viability of the strains used until they reach the small intestine of piglets. On the other hand, it is known that different species of bacteria are subject to adverse conditions in the gastrointestinal tract, and when a exogenous microorganism is fed as a probiotic and enters the gastrointestinal tract, it needs to compete with the existing microbiota ecosystem 43. Thus, in some cases, unfavorable circumstances may end up hindering the proliferation of the microorganism used as a probiotic 44.
The blend of functional oils kept the Muribaculaceae family (phylum Bacteroidetes) stable when compared to the other treatments. Bacteria in this group have been positively related to the regulation of genes for carbohydrate metabolism in mice 45. Muribaculaceae members may be involved in the fermentation of starch into propionate, and its composition is an important predictor of higher concentrations of short-chain fatty acids in healthy intestinal microbiota of the animals 46.
In contrast, the Probiotic provided a greater relative abundance of Veillonellaceae (4%) compared to the functional oils group (2%), but not compared to the control (5%). This family is directly involved in metabolic functions related to proteins and enzymes essential to the host 47.
Shigella spp. and Escherichia coli are closely related and, although they have some differences, they are considered unique genome species. Shigella spp. are among the most important enteric pathogens that cause bacillary dysentery worldwide, especially in humans 48. As observed in Enterobacteriaceae, both additives were effective in inhibiting this genus, with the blend providing slightly less relative abundance. In this same sense, using the E. coli culture technique, it significantly reduced (P < 0.05) E. Coli in the feces of piglets treated with the blend, a result that agrees with those observed by 22. These authors showed that supplementation of 100 ppm of functional oils based on thymol and cinnamaldehyde to the control diet reduced the E. coli count in the feces of weaned piglets.
The reason for these effects may be associated with the antimicrobial activity of phytogenics, demonstrated in vitro by 42. The authors evaluated a mixture based on thymol and cinnamaldehyde and observed its ability to damage the cell membrane and alter the morphology of E. coli and S. aureus pathogenic cells. Similarly, 49 evaluated the supplementation of six essential oils in vivo (including thymol, carvacrol, and eugenol) in piglets challenged by enteropathogenic species (E. coli, Salmonella spp and C. perfringens), reporting antimicrobial activity of these essential oils against at least one of these species. The intermediate results of the Probiotic group agree with other studies 21,44, which also reported the ability of the Probiotic supplementation (L. acidophilus, Pediococcus acidilactici) to reduce fecal E. coli in weaned piglets.
Furthermore, it was possible to observe a reduction of Campylobacter by the supplementation of the blend of functional oils. Campylobacter is the predominant bacterial agent in diarrheal piglets, reducing the relative abundance of bacterial species of the classes Bacteroidia and Clostridia. Both ferment the non-digestible carbohydrate. This reduction results in less production of short-chain fatty acids, which are the main metabolites of the intestinal microbiota, and which could promote barrier function and maintain a healthy and slightly acidic environment in the colon 42.
The erythrocyte, hematocrit, hemoglobin and platelet values of all treatments varied within the reference intervals for young piglets, as recommended by 50. This indicates that the animals were, in general, in good health and not anemic. Similarly, 47 found no significant effect of supplementing 40 ppm of functional Oregano oils on the hematological status of weaned piglets.
The difference in leukocytes circulating in pigs may be associated with two specific factors: inflammatory state or stress state, caused during the weaning period 51. In a study with rats under stress, the levels of circulating inflammatory leukocytes increased by directly stimulating the proliferation of hematopoietic stem cells 52.. Similarly, it has been reported that some types of stressors have increased the total leukocyte count and the proliferation of T cells in pigs 53.
In another study, evaluating hematological parameters in piglets challenged by Salmonella, 54 found no significant differences in Salmonella concentrations in the animals' feces, and suggested that variations in hematological parameters, in that study, were more related to the state of stress than to actual infection by this pathogen. On the other hand, the increase in the percentage of lymphocytes in the blood in piglets challenged by enterotoxigenic Escherichia coli17 was associated with a change in the inflammatory state of these animals, due to the challenge. Opposite results weres observed in this study for the blend of functional oil group, suggesting that it can mitigate E. coli infection.
The commercial blend of functional oils based on cashew nut shell liquid and castor oil improved the performance of piglets weaned during the nursery period. The animals that received probiotics presented intermediate performance and the piglets that did not receive either additive performed worse. The use of functional oils reduced the concentration of Escherichia coli in piglet feces at 50 days of age, demonstrating a modulating effect on the intestinal microbiota of newly weaned piglets.