The beneficial effect of feeding probiotics to improve the health and productivity of feedlot cattle on high-grain diets has been well established7,32. Different lactic acid bacteria (LAB) previously selected as probiotics30,31 were used individually and/or as mixed cultures to supplement feedlot cattle rations in which antimicrobial treatment with the ionophore monensin was administered to animals. Recently, HTS allowed culture-independent metagenomic approaches to access the complete bacterial repertoire within fecal samples. Although the changes in feedlot cattle feces associated with different management practices and health statuses have been widely described, scarce information is available regarding changes occurring in the fecal microbiota structure and population functionality during probiotic administration. In this study, dietary supplementation with three probiotic strains, both individually and in combination, was investigated. Fecal samples from feedlot cattle shared similar community membership and structure regardless of the probiotic group administered. Indeed, it was recently shown that shared environments have a greater impact on shaping the microbiota of individuals than host genetics33. The results from this study agree with the lack of significant differences in alpha diversity detected among treatments when Saccharomyces cerevisiae fermented product, L. casei + L. plantarum, B. subtilis and E. faecium alone or combined with Cl. butyricum were fed to steers, dairy cows, broilers and piglets, respectively34–38. In addition, the distinct clustering patterns that separated the fecal microbiota on E-40 from those on E-104 and E-163 were correlated with the significant differences in beta diversity following supplementation with E. faecium and Cl. butyricum to postweaning pigs38. Thus, a greater influence of administration time compared to that of probiotic groups on microbial diversity of fecal samples was observed in this study.
In line with previous studies on the fecal microbiome from meat animals, a small number of phyla account for the majority of the intestinal microbiota. By applying HTS, fecal samples collected from feedlot cattle administered five probiotic groups provided a detailed view of the fecal microbiome. The dominant phyla found irrespective of probiotic group administration were Firmicutes and Actinobacteria, followed by Bacteroidetes and Proteobacteria, which varied in the number of families and genera among probiotic groups. In this study, probiotic treatments showed a low impact on the microbiome composition among treatments; however, dietary supplementation with probiotics has been reported to alter the gut and fecal microbiota of postweaning pigs and broiler chickens35,38−40. The high dominance of Firmicutes in feedlot feces coincided with those reported for feedlot beef24,26,41−43 and dairy cattle27,44. In this study, families Clostridiaceae, Turibacteraceae, Lactobacillaceae and Ruminococcaceae were predominantly represented in probiotic-administered fecal samples, in agreement with that previously described for the fecal microbiota of feedlot cattle fed high-energy diets13,41,42. Indeed, the relative abundance of Clostridiaceae/Clostridium can influence both positively and negatively the animal host, which is associated with the involved species. As butyric acid producers and beneficially cellulose digestion enhancers, the presence of Cl. tyrobutyricum and Cl. butyricum agrees with those reported in bovine gut and feces46,47. On the other hand, Cl. difficile found in probiotic-treated samples correlated with the high prevalence reported in beef cattle at slaughter, highlighting food contamination potential48,49. In accordance with abundances in the core microbiome and LEfSe analysis, a bloom of Turicibacteraceae from T0 to T3 in all fecal samples was found. Turicibacter has been detected as the dominant genus in the GIT of broilers, cattle rumen and feces27,35,41,50, particularly when high-grain diets were fed to ruminants43,45. A high abundance of Turicibacter sanguinis across all fecal samples was shown; however, it was higher in fecal samples after probiotic C (CRL2069) treatment at T3. A contradictory presumption as a pathogen for this genus was reported51; hence, the potential negative effects on cattle fed grain-rich diets health/performance need to be investigated. In coincidence with LEfSe analysis, the high initial abundance and the subsequent reduction of Lactobacillaceae in feedlot fecal samples agrees with the high predominance during the preweaned period of calves, which decreased over time due to weaning and milk feeding reduction22,23,52. However, variable increases in Enterococcus, Pediococcus and Streptococcus in probiotic-treated fecal samples were observed. In agreement, most LAB found in feedlot fecal samples were also recovered from bovine gut and/or feces49,53. Even at lower abundance, Ruminococcaceae, Lachnospiraceae, Mogibaceriaceae and Leuconostoccaceae were found in all feedlot cattle fecal samples at T3. These bacterial successions suggested that both diet and gut development drive changes in the bacterial composition during life54. Consistent with Rice et al., the presence of R. bromii in the feces of cattle fed wet-distilled grain diets was reported, with a key role in recalcitrant starch in the gut. Moreover, a beneficial in vitro interaction was observed between R. bromii and R. gnavus55, with R. gnavus being able to benefit from starch degradation products released by R. bromii. Within Ruminococcaceae, the presence of F. praunitzii identified from probiotic-treated fecal samples was reported to correlate with health and weight gain in ruminants, exerting intestinal anti-inflammatory effects52,56 In addition, a variable Lachnospiraceae to Christensenellaceae ratio was detected in all fecal samples, and their presence agrees with those found in the fecal and GIT microbiomes of ruminant and nonruminant herbivorous livestock, particularly high feed efficiency steers, dairy cattle and high-grain fed lambs27,44,56−59. These two families play an important role in starch and fiber gut degradation and are commonly associated with animal and human health22,57,60. Moreover, even when the asaccharolytic Mogibacteriaceae family was found in all fecal samples and negatively correlated with the administration time, its function in the ruminant gut is scarcely known56.
As the second predominant phylum, the presence of Actinobacteria is consistent with previous reports for young mammals as healthy infants and calves22,61. However, these findings disagree with other reports for postweaning calves, beef and dairy cattle fecal microbiota, in which Bacteroidetes was the second dominant phylum23,26,27,41. The reduction of Actinobacteria in cattle fecal samples indicated that its presence is age-dependent, being higher during the first weeks of calf life22,24,52,62. Within this phylum, Bifidobacteriaceae and Coriobacteriaceae exhibited the highest relative abundance, the former being the most prevalent. The Bifidobacterium genus, even negatively correlated with the administration time in this study, has a pivotal role in complex carbohydrate metabolism and the maintenance of gut homeostasis; these positive features in the gut have been associated with good health of the host63,64. Species of Bifidobacterium found here were reported to be widely distributed as commensals in the gut and feces of humans and animals65–67. In addition, Coriobacteriaceae abundance agrees with the high relative abundance reported for pasture-fed dairy cows compared to grains fed diets43,44. This family plays important functions in the gut, such as the conversion of bile salts and phytoestrogens as well as the activation of dietary polyphenols; the production of homologs of vitamin K2 also seems to be beneficial68. Changes in the relative abundance of Firmicutes and Actinobacteria phyla in fecal samples reflect the gradual adaptation of calves’ GIT, first to milk and later to solid feed consumption. In the feedlot industry, arriving young steers experienced changes in the diet from forage (silage/hay) to an increasing grain concentration diet (cracked corn, 16 to 78%; soybean expeller, 8 to 10%). Starch abundance and digestibility in grain-fed cattle compared to fiber in forage diets will induce different microbial compositions in fecal bacterial structure and phylum subpopulations responding differently. As reported by Shanks et al., across a starch gradient, Firmicutes changed in composition and decreased in abundance, while no changes in relative abundance were shown for Actinobacteria, but their composition did. In addition, a lower abundance of Corynebacteriaceae was also retrieved from steer faeces, and Corynebacterium (Co.) marinum, Co. maris and Co. stationis were present in control and probiotic-treated samples at T2 and T3. This Actinobacteria genus was reported from perinatal intestinal and cow udder microbiota, indicating the presence of dysbiosis and lower feed efficiency beef cattle69–71. In this study, the Firmicutes to Actinobacteria ratio after probiotic administration decreased toward the end of the fattening cycle, and the highest richness of Actinobacteria was found for the control and probiotic groups at T2 and T3.
In contrast, the relative abundance of the phylum Bacteroidetes as a third population exhibited an increased pattern in probiotic-administered fecal samples at T3. In coincidence with41, this phylum tended to increase in abundance paralleling the grain proportion in the diet. In previous studies, it was reported as the second population following Firmicutes, having a high relative abundance in calves24,62,72 and high grain-fed beef cattle42,43. Even when families from this phylum were not part of the fecal core microbiome, S24-7 or Muribaculaceae family73 and unclassified Bacteroidetes showed the highest richness in treated fecal samples; probiotic groups A and D had the greatest relative abundance at T3. The Prevotella genus detected in much lower abundance agrees with that found in feces from beef cattle fed high-grain diets, which was reported to aid the host in the digestion of complex carbohydrates such starch41–43, 74; in particular, Prevotella copri identified here has been associated with cattle fed high-energy diets56. In coincidence with the scarce recovery of Prevotella in the presence of probiotics, a reduction of this genus after active dry yeast supplementation to lactating dairy cows fed a high-grain diet was reported75.
Furthermore, the Proteobacteria phylum represented in this study by Enterobacteriaceae/Enterobacter showed the highest abundance for the A and D probiotic-treated samples at T3. The presence of Gammaproteobacteria members coincided with those reported for feces of grains fed cattle41,42. It is known that a Proteobacteria increase in the gut may be indicative of dysbiosis facilitating inflammation or invasion by pathogens76. Unsurprisingly, fecal samples after probiotic group C (L. mucosae CRL2069) showed the lowest abundance, coinciding with the immunobiotic ability of this probiotic strain protecting bovine host against TLR-mediated intestinal inflammatory damage, as was recently described16. In addition, the decreased abundance of the Tenericutes phylum at T3 in feedlot fecal samples correlates with that reported for beef and dairy cattle feces27,28,41,77. Bacteria lacking peptidoglycan cell walls and typically parasites or commensals of eukaryotic hosts are considered potential pathogens, and their function in the gut remains unclear. Other phyla in low abundance included Spirochaetes and TM7 and were found in all fecal samples, while Verrucomicrobia, Chloroflexi and Cyanobacteria were not. Even with a poor physiology understanding, the TM7 phylum appears to be ubiquitous in the GIT of mammals and is reported as part of the bacterial community of lactating dairy cows and beef cattle69,77.
Identifying a core microbiome is the first step to predict its response to perturbations and to define a microbial community that will guide its manipulation for desired outcome achievement78. The presence of core taxa across fecal samples indicates that these microorganisms perform metabolic functions universal to the collective cattle fecal microbiome. In this study, although sequences were assigned to numerous taxa, only a small number of them (2 phyla, 5 orders, 11 families, and 27 genera) were detected across all cattle fecal samples. Firmicutes and Actinobacteria represented two of the ten phyla found across all fecal samples of feedlot cattle administered probiotics, while no taxa from Bacteroidetes were involved. Nevertheless, Bacteroidetes was reported as a core phylum in the gut of bovines fed high-energy diets. Indeed, in the absence of probiotics, the Prevotella genus was consistently found as part of the core microbiome in the feces of beef cattle fed high-energy diets41–43. In this study, probiotic administration to feedlot cattle seemed to stimulate the presence of “health-associated” bacterial species such as those from Ruminococcaceae, Lachnospiraceae and Bifidobacteriaceae identified among the core microbiome. These results are in accordance with the modulatory effect of feed additives such as phytobiotics and live/autolyzed yeast used in cows fed high-grain diets, which increased the abundance of gram-positive bacteria and decreased that of gram-negative bacteria8. Moreover, oral administration of probiotics (L. plantarum) to dairy calves was found to affect the rumen bacterial community with a decrease in the numbers of cellulolytic bacteria79. As an important component of the diet, it is worth noting that the presence of ionophore monensin could also be involved in fecal microbiome changes, since it has been shown to alter digestive microbiota in cattle fed a forage diet of members of the phylum Bacteroidetes80. However, it seems unlikely that the presence or absence of an ionophore is the sole explanation for shifts in the population compositions of Firmicutes and Bacteroidetes. Other factors, such as cattle breeding, environment, feeding practices and management, may also account for the succession in phyla abundance found in this study.
It is known that the cattle GIT microbiome fulfills many physiological functions that are lacking in the host; thus, they are considered essential to cattle life. To determine the potential functions of the microbiota identified from fecal samples, PICRUSt2 was used to infer putative abilities by prediction of functional genes that are typically associated with different taxa. The main metabolic pathways found in this study are consistent with the role of these functions in microbial life81. Abundances affected by the different experimental periods of probiotic administration were compared, since a higher impact on fecal microbiome structure was before determined. Pairwise comparisons among administration periods revealed significant differences in bacterial functions; the E-40 period exhibited the highest number of metabolic pathways. Carbohydrate degradation (mannan, fucose, glycerol, hexitol) pathways agree with the high forage rich in cellulose and hemicellulose (> 60%) fed to feedlot cattle; the early high presence of Firmicutes/Clostridiales in the fecal microbiome would account for these activities. Similar results were reported for the functional capacity of the gut/fecal microbiota of ruminants, swine and broilers27,81−83. In addition, biosynthetic and derivative pathways related to amino acids and protein metabolisms detected were higher in the E-40 experimental period. As recently reported85, Lachnospiraceae, Ruminococcaceae and mostly Clostridiaceae are commensal with a high role in the digestion of carbohydrates and proteins, which agrees with their richness as microbiome core members. Species from these families were associated with better feed efficiency or weight average daily gain, as fermenters of a range of complex plant-derived polysaccharides with production of short-chain fatty acids, particularly butyrate, that will be used as energy source83,84. As a result, it may be suggested that 40 days of probiotic administration to feedlot cattle is optimal for the activation of beneficial metabolic pathways by stimulating the growth of health-associated bacteria. In coincidence, 40 days of administration of probiotic group A was also shown to be enough for a positive modulation of the microbiome according to the similar relative abundance at the genus level obtained from 40 to 163 days.