Saladrigas‑García et al. Animal Microbiome https://doi.org/10.1186/s42523-022-00221-9 (2022) 4:68 Animal Microbiome RESEARCH Open Access An insight into the commercial piglet’s microbial gut colonization: from birth towards weaning Mireia Saladrigas‑García1, Mario Durán2, Matilde D’Angelo1, Jaume Coma2, José Francisco Pérez1 and Susana María Martín‑Orúe1* Abstract Background: The establishment of the gut microbiota can be influenced by several perinatal factors, including, most importantly, the maternal microbiota. Moreover, early-life environmental variation affects gut microbial colonization and the intestinal health of offspring throughout life. The present study aimed to explore the development of piglet gut microbiota from birth to weaning in the commercial practice and also to assess how different farm environments could condition this process. Although it is possible to find in the literature other studies with similar objectives this work probably represents one of the few studies that make a systematic evaluation of such differential factors under a real scenario. To achieve this objective, we performed two trials. In a first Trial, we selected 2 farms in which we performed an intensive sampling (5 samples /animal) to characterize the gut colonization pattern during the first days of life and to identify the time window with the greatest impact. Both farms differed in their health status and the use of antimicrobials in the piglets. In a second Trial, we selected 4 additional farms with variable rearing conditions and were obtained with swabs and DNA was extracted by using the PSP® Spin Stool DNA Kit and sequencing of the 16S a distinctive use of antimicrobials in the sows with a simplified sampling pattern (2 samples/animal). Faecal samples rRNA gene (V3-V4 region) performed by Illumina MiSeq Platform. Results: The present study contributes to a better understanding of microbiome development during the transition from birth to weaning in commercial conditions. Alpha diversity was strongly affected by age, with an increased rich‑ ness of species through time. Beta diversity decreased after weaning, suggesting a convergent evolvement among individuals. We pinpointed the early intestinal colonizers belonging to Bacteroides, Escherichia-Shigella, Clostridium sensu stricto 1, and Fusobacterium genera. During lactation(d7-d21 of life), the higher relative abundances of Bac- teroides and Lactobacillus genera were correlated with a milk-oriented microbiome. As the piglets aged and after weaning (d36 of life), increasing abundances of genera such as Prevotella, Butyricimonas, Christensenellaceae R-7 group, Dorea, Phascolarctobacterium, Rikenellaceae RC9 gut group, Subdoligranulum, and Ruminococcaceae UCG-002 were observed. These changes indicate the adaptation of the piglets to a cereal-based diet rich in oligosaccharides and starch. Our results also show that the farm can have a significant impact in such a process, evidencing the influence of different environments and rearing systems on the gut microbiota development of the young piglet. Differences *Correspondence: susana.martin@uab.cat 1 Servicio de Nutrición Y Bienestar Animal. Departamento de Ciencia Animal Y de los Alimentos, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. Saladrigas‑García et al. Animal Microbiome (2022) 4:68 Page 2 of 23 between farms were more noticeable after weaning than during lactation with changes in alpha and beta biodiversity and specific taxa. The analysis of such differences suggests that piglets receiving intramuscular amoxicillin (days 2–5 of life) and being offered an acidifying rehydrating solution (Alpha farm in Trial 1) have a greater alpha diversity and more abundant Lactobacillus population. Moreover, the only farm that did not offer any rehydrating solution (Foxtrot farm in Trial 2) showed a lower alpha diversity (day 2 of life) and increased abundance of Enterobacteriaceae (both at 2 and 21 days). The use of in-feed antibiotics in the sows was also associated with structural changes in the piglets’ gut ecosystem although without changes in richness or diversity. Significant shifts could be registered in different micro‑ bial groups, particularly lower abundances of Fusobacterium in those piglets from medicated sows. Conclusions: In conclusion, during the first weeks of life, the pig microbiota showed a relevant succession of micro‑ bial groups towards a more homogeneous and stable ecosystem better adapted to the solid dry feed. In this relevant early-age process, the rearing conditions, the farm environment, and particularly the antimicrobial use in piglets and mothers determine changes that could have a relevant impact on gut microbiota maturation. More research is needed to elucidate the relative impact of these farm-induced early life-long changes in the growing pig. Keywords: Pig, Suckling piglet, Weaning, Microbiota, Colonization, Lactation, Gut health, Commercial farm Background host phenotype and its impact on animal response and The process of microbial colonization of the gut after performance. birth plays an important role in the development of In commercial pig husbandry, weaning is an abrupt the neonatal immune system of mammals with impli- event comprising a dietary shift from sow milk to solid- cations during their whole life [1]. Immediately after feed-based diets, which poses a challenge to piglets dur- birth, environmental and maternal bacteria, includ- ing early-life development. During the pre-weaning ing colonization via the vagina, nipple surface, and phase, microbiome composition is dominated by a milk- milk, quickly colonize the offspring gut and establish oriented microbiome composed of families like Bacte- the initial microbiota of the piglet [2–4]. The intestinal roidaceae and Lactobacillaceae [16, 17], which rapidly microbiota protects against colonization by pathogens changes after weaning when a solid cereal-based diet is through bacterial competition and interaction [5]. The introduced. For instance, butyrate-producing genera such disruption of the healthy microbial community dur- as Prevotella, having a very low abundance in suckling ing the neonatal period may lead to the overgrowth of piglets, dramatically increase post-weaning due to the indigenous pathobionts and induction of pro-inflam- availability of complex oligosaccharides and polysaccha- matory status [6, 7]. It has been shown that stress, diet, rides in the feed [16, 18, 19]. The rapidly changing micro- management practices, and antimicrobial compounds biome of the young piglets seems to increase in diversity during the early-life period may induce a long-lasting and richness along with the suckling phase and gradually impact on the establishment of gut microbiota, disease stabilizes postweaning [16, 17, 20–22]. However, despite susceptibility, and growth performances of offspring the recent advances in the knowledge of the development pigs [7–14]. This is especially relevant in swine pro- of intestinal microbiota in the young pig, there is still a duction in which differences in husbandry and farm lack of knowledge regarding how this pattern can be environment along the first days of life, could have a influenced by the farms’ management practices and the long-lasting impact on animal health and productive microbiological environment in which piglets are raised. outcome. For instance, some researchers have reported Considering the great importance of the early gut how different exposure to stress or the use of antibiot- microbiota development for pig future health and pro- ics can determine changes in the gut microbial coloni- ductive life, the objective of this work was, therefore, to zation of piglets 8 days after birth with implications in determine the succession of microbial colonization that the immune development [11]. Evidence has also been occurs in those piglets reared in commercial conditions published defining differences in the faecal microbiota and to identify possible pattern changes associated to of piglets as early as 7 days of life, determining their different management guidelines and particularly to the susceptibility to suffering post-weaning diarrhoea four use of antimicrobials. For that purpose, we performed weeks later [15], emphasizing the potential of the early two trials. In Trial 1, we selected 2 farms differing in microbiota establishment on the development of the their health status and the use of antimicrobials in the immune response. It is, therefore, crucial to accurately piglets performing an intensive sampling (5 samples / determine the early-life development of the gut micro- animal). In Trial 2, we selected 4 additional farms with biome to eventually unravel microbiome effects on Saladrigas‑García et al. Animal Microbiome (2022) 4:68 Page 3 of 23 a distinctive use of in-feed antibiotics in the sows and a were fed a feed without ZnO in all cases. In each farm, simplified sampling pattern (2 samples/animal). 10 litters were randomly selected, and one average piglet per litter was sampled. The same piglet was sampled on Methods d2, d7, d14, and d21 of lactation and d14 after weaning Animal ethics and experimental design (d36 of life), obtaining a total of 100 samples for Trial 1. The experimental work was approved by the Animal However, in Alpha farm, the faecal sampling after wean- Ethics Committee at the Autonomous University of ing had to be anticipated due to an imminent antibiotic Barcelona. treatment of the piglets (d7 after weaning and d29 of life) A longitudinal analysis of the development of the fae- because of a diarrhoea outbreak. cal microbiota of piglets reared under commercial con- For the second Trial of the study, four farms were ditions was carried out. A total of 6 farms (S1 and S2) selected (Charlie, Delta, Echo and Foxtrot) trying also to from the same vertical integrator were selected, two analyse the possible impact of the use of antibiotics in the were included in a first Trial and four in a second Trial. mothers’ diet. In two of them (Charlie and Delta) sows Each trial was performed at a different time of the year. were fed NMF and in the other two (Echo and Foxtrot) The first Trial included an intensive sampling during the sows received medicated feed with lincomycin (600 ppm) piglets’ first month of life approximately (5 time -points (ABF). Piglets from all farms were offered creep feed for each animal on days 2, 7, 14, 21, and 36 of life), and without ZnO and piglets from Charlie, Delta and Echo of life (Hidravall®, MEVET S.A.U, Lleida, Spain). In each the second Trial a simplified sampling pattern (2 time- farms a rehydrating oral solution during the first week points for each animal on days 2 and 21). Stool samples were collected from the piglets by rectal swab and stored farm 15 litters were randomly selected, sampling one pig- at room temperature until analysed. Piglets were a com- let per litter. The same piglet was sampled on d2 and d21. mercial crossbreed Piétrain × (Landrace × Large White) Due to some casualties on d21 (by non-infections causes coming from multiparous sows. General information such as crushing, low viability or starvation) ultimately about the six farms involved in the study can be found in 102 paired samples (d2 + d21 from the same animal) were Table 1. analysed. Information regarding the composition of lac- The two farms included in the first Trial (Alpha and tation diets (NMF and ABF) is provided in Additional Bravo farms) were selected based on their different health file 1: Table S1. status and use of antimicrobials. Whereas Alpha could be considered a high-standard farm with a low incidence of Faecal DNA extraction and 16S rRNA sequencing pathologies, Bravo frequently coursed episodes of pleu- Stool samples were taken with the Stool Collection Tubes ropneumonia (Actinobacillus pleuropneumonia, APP) with DNA Stabilizer (STRATEC Molecular GmbH, Ber- stool-preserved sample using the PSP® Spin Stool DNA and swine dysentery (Brachyspira hyodysenteriae). Alpha lin, Germany). DNA was extracted from 1.4 mL of each was involved in an antibiotic reduction program and pig- lets received an intramuscular dose of amoxicillin (15 mg Kit (STRATEC Molecular GmbH, Berlin, Germany) amoxicillin/kg BW) between their second and fifth day according to the manufacturer’s instructions following acidifying solution the first week (Hidracid®, MEVET of life; moreover, they were given an oral rehydrating and the optimization steps for bacterial DNA enrichment. DNA concentration and purity were verified with the S.A.U, Lleida, Spain) including dextrose, sodium chlo- NanoDrop ND-1000 spectrophotometer (NanoDrop put sequencing of the faecal microbiota, the MiSeq® ride, glycerine, monobasic potassium phosphate, mono, Technologies, Wilmington, DE, USA.). For high-through- di and triglycerides of medium-chain fatty acids (C4, C8, and C10), potassium chloride and organic acids (formic Reagent Kit V2 (500 cycles) (Illumina, San Diego, CA, acid, sodium formate, propionic acid, sodium citrate). In USA) was used and the V3-V4 region of 16S rRNA was Bravo, piglets did not receive any antibiotic treatment, targeted. All subsequent steps were performed on the although the mothers received a tulathromycin injection MiSeq Illumina instrument. on day 16 post-partum (2.5 mg tulathromycin/kg BW). the 1st week of life (Hidravall®, MEVET S.A.U, Lleida, The piglets were also given an oral rehydrating solution Bioinformatics and statistical analysis Raw sequencing reads (Fastq files) were independently Spain) including dextrose, sodium chloride, monopotas- processed, aligned and categorized using Divisive sium phosphate, and potassium chloride. In both farms, Amplicon Denoising Algorithm 2 (DADA2) [23], which piglets were offered creep feed without zinc oxide. Moth- was run as an R script (in R v.4.0.2) using its R package ers received a lactating non-medicated feed (NMF). (dada2 v.1.16.0). Sequence reads were filtered using the After weaning the piglets were mixed according to the recommended DADA2 parameters (that is, an expected usual management procedures in each farm. The piglets error threshold of 2) and chimaeras were removed Table 1 Additional general information about each one of the six farms involved in the study Farm ID Trial 1 Trial 2 Alpha Bravo Charlie Delta Echo Foxtrot Number of ­sows1 1330 (10) 5144 (10) 3240 (15) 1800 (15) 518 (15) 2790 (15) Production schedule Weekly Weekly Weekly Weekly 4-week period Weekly Biosafety level High High High High High High Closed-cycle farm No No No No No No Farm involved in an AB Yes No No No No No reduction program Saladrigas‑García et al. Animal Microbiome Sow vaccination program PRRS, porcine parvo‑ PRRS, porcine parvovirus, PRRS, porcine parvovirus, PRRS, porcine parvovirus, PRRS, porcine parvovirus, PRRS, porcine parvovirus, virus, swine erysipelas, swine erysipelas, Aujeszky, swine erysipelas, Aujeszky swine erysipelas, Aujeszky swine erysipelas, Aujeszky, swine erysipelas, Aujeszky, Aujeszky, Actinobacillus APP (toxoid), Mycoplasma and colibacillosis and colibacillosis colibacillosis and rotavirus swine influenza, colibacillo‑ pleuropneumonia (APP), hyopneumoniae, swine in gilts sis and porcine circovirus colibacillosis and rotavirus influenza, colibacillo‑ in gilts (2022) 4:68 sis + clostridiosis, rotavirus in gilts and swine dysen‑ tery autovaccine Recurring sanitary prob‑ No APP and B. hyodysenteriae No No B. hyodysenteriae B. hyodysenteriae lems Piglet processing at birth Tail docking, iron admin‑ Tail docking, iron admin‑ Tail docking, iron admin‑ Tail docking, iron admin‑ Tail docking, iron admin‑ Tail docking, iron admin‑ istration by injection, istration by injection and istration by injection, istration by injection, istration by injection, istration by injection, oral administration of oral administration of oral administration of oral administration of oral administration of oral administration of coccidiostat and antibiotic coccidiostat coccidiostat and antibiotic coccidiostat and antibiotic coccidiostat and antibiotic coccidiostat and antibiotic treatment (injected) treatment (injected) treatment (injected) treatment (injected) treatment (injected) Regular use of creep- Yes Yes Yes Yes Yes Yes feeding Use of AB in creep-feeding No No No No No No (including ZnO) Regular use of re- Yes Yes Yes Yes Yes No hydrating solution during lactation Piglet age at weaning 21 days 21 days 28 days 21 days 21 days 21 days Cross fostering and litter Nursing mothers at 24 h. Equalization of litters Minimal movements Minimal movements Minimal movements Minimal movements management at birth Minimal movements trying to minimize movements. Piglets are collected only during the 1st week Medicated maternal feed No, NMF diet No, NMF ­diet2 No, NMF diet No, NMF diet Yes, ABF ­diet3 Yes, ABF ­diet2 during lactation 1 The numbers in parentheses indicate the number of sows selected for the study 2 Bravo farm sows routinely received a tulathromycin injection on day 16 postpartum (2.5 mg tulathromycin/kg BW) 3 ABF diet consisted of the same NMF diet supplemented with 600 ppm of lincomycin Page 4 of 23 Saladrigas‑García et al. Animal Microbiome (2022) 4:68 Page 5 of 23 (“removeBimeraDenovo” command). Afterwards, compared to Alpha farm (p < 0.001 and p = 0.007 for spe- sequences were processed into Amplicon Sequences Var- cies richness and reads, respectively). iants (ASV) at 99% of identity. ASV were classified to the Regarding changes in the ecosystem structure with lowest possible taxonomic level based using the SILVA age and environmental factors, PERMANOVA multi- reference database (v138) provided by the SILVA web variate analysis showed that both, farm and animal age, service [24]. The diversity patterns within the ASV table showed significant effects in shaping the gut communi- were analysed using a custom bioinformatics pipeline ties (p < 0.001). The non-metric multidimensional scaling implemented in R 4.0.2 (http://​www.r-​proje​ct.​org). Sup- (NMDS) based on the Bray–Curtis distance, showed five port for DADA2 in R was achieved through the phyloseq clusters that matched with piglet age (Fig. 1). The diver- package (v.1.32.0; available at https://​joey7​11.​github.​io/​ sity indices also revealed important differences related phylo​seq/) [25]. The alpha diversity metrics were calcu- to farms and ages. Following the richness results, greater lated using the phyloseq “estimate_richness” function alpha diversity was observed in Bravo farm and with the from the rarefied ASV tables and using the microbiome increasing age of piglets (p < 0.001), as presented in Addi- package (v.1.10.0) [26]. The observed species, the Chao1 tional file 1: Table S2. In addition, measured by the ANO- index, the Simpson and inverse Simpson metrics and the SIM Bray–Curtis dissimilarities matrix, beta diversity Shannon diversity measures were estimated. For beta also showed increased values with age (p = 0.001) sug- diversity, measurements were calculated using the Whit- gesting that as the piglets grow and the gut community taker index [27] and the betadisper () function of the becomes more diverse, the divergence among animals vegan package (v.2.5.6) [28] using relative abundances. increases. To compare any differential effects, an ANOVA analy- To better analyse the impact of the farm, a comple- sis was performed for alpha richness and diversity. The mentary analysis was performed by sampling day. PER- Non-metric multidimensional scaling (NMDS), analy- MANOVA within each sampled age (Additional file 1. sis of similarities (ANOSIM), permutational analysis of Figure S1) showed a clear effect of the farm on commu- variance (PERMANOVA) and the method of grouping of nity composition on all sampling days (p < 0.01). Along unweighted pairs with hierarchical grouping of arithme- lactation differences between farms in alpha diversity tic mean (UPGMA), all of them based on the distance of (Additional file 1. Table S3) were significant at days 7 and Bray–Curtis, were carried out to test the significance of 14 of life, showing Bravo farm higher values (p < 0.05 for differences in overall microbial composition. The normal- all indices). However, no differences were found on day ization of the raw counts was performed using cumula- 21, probably due to the establishment of a more mature tive sum scaling (CSS) [29] and the differential abundance microbiota. Finally, after weaning a large impact of the analysis was performed following the metagenomeSeq farm of origin was observed (PERMANOVA, p < 0.001), package (v.1.30.0) [30]. The taxa were aggregated at the in part due to the advanced sampling in the Alpha farm phylum, family and genus levels and expressed as com- (7 days earlier than in the Bravo farm). Differences in the positional data. Environmental (farm or administration alpha and beta diversities between farms were also seen of antibiotics to the mother (Trial 2)) and host-covariates after weaning (p < 0.001). (age) were all considered factors that might modulate the diversity, structure and profile of the microbial commu- Changes in taxonomic groups nities. All p-values from multiple testing were corrected The temporal progression in the relative abundances of with a false discovery rate (FDR) according to the pro- the main families is represented in Table 2 and Fig. 2. In cedure by Benjamini–Hochberg [31]. Differences were the sampling closest to birth (day 2 of life), the microbi- expressed as significant if adjusted p ≤ 0.05 or tendencies ota of the piglets was composed of bacteria belonging to if 0.05 < p < 0.1. Results are presented as the mean ± the the phyla Firmicutes (36.98%), Proteobacteria (29.68%), standard error (SE). Fusobacteria (18.34%) and Bacteroidetes (14.30%). Within the Proteobacteria, the main families observed Results were Enterobacteriaceae (24.62%), of which 10.30% were Trial 1: intensive sampling in two farms with different identified as Escherichia/Shigella, and Pasteurellaceae health statuses (4.50%). The phylum Bacteroidetes was represented by its Changes in microbiota structure and biodiversity main genus Bacteroides (11.89%), while the phylum Fir- An average of 55,190 ± 6,265 amplicon sequence vari- micutes was represented by a greater variety of families ants (ASV) were obtained per sample, for a total of 100 and genera, such as Clostridiaceae (18.56%), represented samples of faecal content, with an increase in species mainly by Clostridium sensu stricto 1 (4.05%), Lachno- richness as the piglets aged (p = 0.002). Higher richness spiraceae (7.87%), Streptococcaceae (3.41%) and Lacto- values and reads were also detected in piglets from Bravo bacillaceae (1.97%), with a great relative abundance of its Saladrigas‑García et al. Animal Microbiome (2022) 4:68 Page 6 of 23 Fig. 1 NMDS of the relative abundances of ASV in Trial 1. Different colours have been used to highlight each sampling day. The farm of origin is indicated with a different shape, round for the Alpha farm and with a 3-pointed star for the Bravo farm. A significant impact shaping the gut communities is produced by age and environmental factors (p < 0.001) with five clusters that match with piglet age. A greater richness is observed in Bravo piglets when compared to Alpha’s (p < 0.001), and with the increasing age of piglets (p < 0.001), suggesting that as the piglets grow and the gut community becomes more diverse, the divergence among animals increases. The significant effect of the farm is observed more clearly when performing the analysis by sampling day (Additional file 1. Figure S1) genus Lactobacillus (4.21%). Finally, the high presence of predominant phyla. The abundance of Fusobacteria bacteria from the Fusobacteriaceae family (18.31%) and dropped to a great extent (4.11% and 2.19% at d14 and its genus Fusobacterium (10.04%) was found very charac- d21, respectively) and Proteobacteria stabilized with a teristic of young piglets (d2 and d7). relative abundance at the phylum level of 10%. At this At one week of life (d7), a large decrease in the rela- stage, there were no major changes in the families or tive abundance of the phylum Proteobacteria (6.89%) was genera present in the microbiota. Moreover, a higher observed, mainly due to the large decrease also observed evenness was observed, as the relative abundances of in the Enterobacteriaceae family (4.67%), despite the taxonomic groups seemed to equal each other in abun- fact that the relative abundance of Escherichia/Shigella dance, with less prevalence of specific genera as in the genus level remained high (12.38%). There was a signifi- first days of life (evenness: 0.88 ± 0.043; 0.91 ± 0.027; cant increase in the relative abundance of Bacteroidetes 0.92 ± 0.024; 0.92 ± 0.036 and 0.95 ± 0.013 for day 2, 7, (35.64%), due to the increase in abundance of genera 14, 21 and WP respectively). Thus, higher abundances such as Prevotella (6.34%), Rikenellaceae RC9 gut group of bacteria belonging to Firmicutes were observed, such (1.94%) and CAG-873 (1.82%). Firmicutes remained as Lachnospiraceae (14.84% and 14.56% at d14 and d21, the predominant phylum (41.25%), although impor- respectively), Oscillospiraceae (7.66% and 7.85%), Rumi- tant changes were observed in its distribution, with nococcaceae (3.81% and 2.75%), Erysipelotrichaceae increases in families such as Lachnospiraceae (12.26%), (1.49) % and 1.59%) and Christensenellaceae (1.37% and Lactobacillaceae (6.19%), Oscillospiraceae (5.40%) and 2.00%). A slight decrease was also observed in the rela- Acidaminococcaceae (3.48%) and a large decrease in the tive abundance of Lactobacillaceae (5.26% and 3.99% Clostridiaceae family (1.03%), despite the fact that the at d14 and d21, respectively), and in the Bacteroidetes relative abundance of the genus Clostridium sensu stricto families, such as Bacteroidaceae (10.07% and 14.97% at 1 remained high (5.21%). Fusobacteria decreased slightly d14 and d21, respectively) and Prevotellaceae (7.42% (11.95%), although at genus level Fusobacterium contin- and 7.29% at d14 and d21, respectively). ued to predominate (10.00%). Finally, after weaning, a greater variety of species On day 14 and day 21, the microbial composition and evenness among them was observed. Firmicutes showed a more similar outcome to each other. Firmi- (43.69%) and Bacteroidetes (31.27%) were kept as pre- cutes and Bacteroidetes established themselves as the dominant phyla, followed by Proteobacteria (15.50%). The Spirochaetes phylum increased significantly in Saladrigas‑García et al. Animal Microbiome (2022) 4:68 Page 7 of 23 Table 2 Relative abundances (%) of the families present in the faecal microbiota of the piglets in a percentage higher than 0.5% d2 d7 d14 d21 WP SEM P-value Bacteroidaceae 10.64 17.28 10.07 14.97 8.18 1.185 0.038 Lachnospiraceae 7.87 12.26 14.84 14.56 8.81 0.648 0.002 Enterobacteriaceae 24.62 4.67 6.44 6.66 8.03 1.272 0.002 Prevotellaceae 3.12 9.71 7.42 7.29 11.87 0.891 0.116 Fusobacteriaceae 18.31 11.04 4.11 2.19 3.51 1.057 0.025 Oscillospiraceae 0.32 5.40 7.66 7.85 8.58 0.625 0.000 Clostridiaceae 18.56 1.03 1.66 1.23 2.59 0.892 0.000 Lactobacillaceae 1.97 6.19 5.26 3.99 3.03 0.513 0.014 no_matcha 0.23 3.25 5.91 4.46 6.53 0.368 0.012 Muribaculaceae 0.04 3.23 5.54 5.27 3.47 0.455 0.000 Rikenellaceae 0.01 2.72 3.46 3.93 3.13 0.325 0.000 Acidaminococcaceae 0.47 3.48 3.46 2.95 2.86 0.221 0.000 Ruminococcaceae 0.20 2.64 3.81 2.75 3.36 0.301 0.000 Streptococcaceae 3.41 1.06 2.05 0.46 1.55 0.303 0.116 Campylobacteraceae 0.14 1.64 2.37 1.50 2.43 0.259 0.000 Spirochaetaceae 0.00 0.82 0.81 0.87 4.73 0.292 0.002 Pasteurellaceae 4.50 0.90 0.70 0.65 0.38 0.244 0.006 Erysipelotrichaceae 0.17 0.95 1.49 1.59 1.82 0.145 0.019 Marinifilaceae 0.16 1.00 1.37 2.23 1.12 0.151 0.000 Christensenellaceae 0.00 0.61 1.37 2.00 1.59 0.176 0.000 Veillonellaceae 1.96 1.34 0.68 0.75 0.39 0.143 0.168 Tannerellaceae 0.22 1.03 0.82 1.17 1.17 0.109 0.004 Selenomonadaceae 0.01 1.13 0.78 1.77 0.36 0.252 0.239 Desulfovibrionaceae 0.17 0.80 0.94 1.27 0.73 0.072 0.000 Enterococcaceae 0.85 0.56 0.97 0.83 0.32 0.167 0.999 Suerellaceae 0.25 0.65 0.52 0.48 0.27 0.055 0.082 Erysipelatoclostridiaceae 0.02 0.38 0.50 0.58 0.61 0.057 0.007 p-2534-18B5_gut_group 0.00 0.15 0.26 0.50 1.10 0.107 0.239 Anaerovoracaceae 0.00 0.43 0.47 0.29 0.72 0.057 0.000 Butyricicoccaceae 0.53 0.45 0.34 0.21 0.26 0.058 0.190 Succinivibrionaceae 0.00 0.36 0.25 0.37 0.72 0.095 0.003 UCG-010 0.00 0.15 0.20 0.28 0.84 0.052 0.065 Akkermansiaceae 0.00 0.29 0.01 0.65 0.40 0.096 0.006 Helicobacteraceae 0.01 0.42 0.06 0.13 0.57 0.083 0.003 Synergistaceae 0.00 0.02 0.16 0.70 0.20 0.053 0.130 Bacteroidales_RF16_group 0.00 0.08 0.08 0.13 0.48 0.048 0.999 a no_match = Not assigned taxa. ■: Firmicutes ■: Bacteroidetes ■: Fusobacteria ■: Proteobacteria ■: Spirochaetes ■: Verrucomicrobiota ■: Synergistetes □: None Results came from 20 piglets sampled from two different farms during the first days of life. Faecal samples were collected on days 2, 7, 14 and 21 of lactation and 14 days after weaning (36 days of life). Results are presented in decreasing order of abundance concerning the general average. The colours represent the phyla to which the families belong. Significant changes are highlighted in bold. Saladrigas‑García et al. Animal Microbiome (2022) 4:68 Page 8 of 23 this period, with a relative percentage of 4.74%. Fuso- due to the higher counts observed in the Enterobacte- bacteria maintained levels similar to those observed riaceae family (p = 0.010) and its genus Escherichia/Shi- before weaning (3.52%). At the family level, an increase gella (p < 0.001); the family Bacteroidaceae (p = 0.043) and was observed with respect to the previous sampling in its genus Bacteroides (p < 0.001) and the various families Prevotellaceae (11.87%) and Spirochaetaceae (4.73%), belonging to Firmicutes: Lachnospiraceae, Streptococcaceae and a decrease in Lachnospiraceae (8.81%). At the and Ruminococcaceae (p = 0.010, p = 0.016, and p = 0.030) genus level, a decrease in Escherichia/Shigella was and their genera Streptococcus (p < 0.001), Lactobacil- observed with respect to the lactating piglets (5.31%). lus (p < 0.010), Lachnoclostridium (p = 0.015) and Dorea The effects of the farm of origin (Alpha or Bravo) in the (p = 0.018), all of them, as stated before, with a higher pres- relative abundances of main families and genera is repre- ence in the piglets from the Bravo farm. sented in Fig. 3. At two weeks of age (d14), the farm factor seems to have The taxonomic groups that showed significant differ- a lesser impact, with some differences between taxonomic ences between farms along the different sampling days groups, characterized mainly by a higher number in the can be found in Additional file 1: Figure S2 consider- piglets belonging to the Bravo farm of families belong- ing only the taxa detected in at least half of the samples. ing to the Firmicutes phylum, such as Streptococcaceae In general terms, it was observed that at day 2 of life and (p = 0.012) and Lachnospiraceae (p = 0.041) and the gen- after weaning, the impact of the farm was greater than at era Lachnoclostridium (p = 0.005) and Dorea (p = 0.015). days 7, 14 and 21 of life, since a higher number of differen- Similarly, higher counts of Bacteroidetes are observed in tial taxa were detected. After weaning, differences among piglets reared on the Bravo farm, coinciding at the family farms were manifested with significant changes in more level with Prevotellaceae (p = 0.019) and its genus NK3B31 than 19 genera. It is interesting to remark the lower num- group (p = 0.037). bers of Enterobacteriaceae and Escherichia/Shigella genera At day 21 of life, just before weaning, a great significance observed in Alpha farm on days 2 and 7 of life (2.9 and 3.4 is observed in the differential abundance of Fusobacteria negative ln change respectively) and the opposite pattern (p = 0.016), with higher counts in the piglets reared on the registered after weaning (3.2 positive ln change compared Bravo farm either at the level of phylum, family (p = 0.019) to Bravo farm). or gender (p = 0.017). Likewise, lower Lactobacillus More specifically and looking for differences between counts were detected in piglets reared on the Alpha farm farms, we observed that at the phylum level and in 2-day- (p = 0.046). old piglets, Proteobacteria were increased in piglets from After weaning great shifts were observed. Fusobac- Bravo farm (p = 0.001). In parallel, the corresponding fam- teriaceae and Enterobacteriaceae, and their respective ily and genus, Enterobacteriaceae and Escherichia/Shigella, genera, Fusobacterium and Escherichia/Shigella, were sig- respectively, were also enriched in Bravo-reared animals nificantly more enriched in piglets reared on the Alpha compared to the Alpha-reared animals (p < 0.001). Bacte- farm (p = 0.010 and p < 0.001 for Fusoacterium and Escheri- roidetes was also found to be increased in piglets from the chia/Shigella, respectively). On the contrary, various fami- Bravo farm (p = 0.008), with the subsequent increase in its lies and genera belonging to the Firmicutes phylum were genus Bacteroides (p < 0.001). Some taxa belonging to the found enriched in the piglets reared on the Bravo farm. Firmicutes phylum were found enriched in piglets from the For instance, within the Lachnospiraceae family, there were Bravo farm, such as the Enterococcaceae family (p < 0.001) higher counts of Coprococcus (p < 0.001), Lachnospiraceae and its genus Enterococcus (p < 0.001), and other genera UCG-004 (p = 0.004), Lachnoclostridium (p = 0.009) and such as Lactobacillus (p < 0.001), Phascolarctobacterium Dorea (p = 0.028). Similarly, in piglets from the Bravo farm, (p < 0.001) and Clostridium sensu stricto 1 (p = 0.003). On higher counts of Streptococcus (p < 0.001), Faecalibacte- the contrary, the Actinobacillus and Dorea genera obtained rium (p < 0.001), Phascolarctobacterium (p = 0.008), and higher counts in the piglets from the Alpha farm (p = 0.002 Lactobacillus (p = 0.021), among others, were observed and p = 0.016, respectively). with respect to the piglets from the Alpha farm. Finally, At one week of life (d7), the three predominant phyla: Fir- the Bacteroidetes phylum was also found to be enriched in micutes, Proteobacteria and Bacteroidetes, obtained higher piglets from the Bravo farm, with higher counts of genera counts in the piglets from the Bravo farm. This could be such as Prevotella (p < 0.001), Prevotellaceae NK3B31 group (See figure on next page.) Fig. 2 Differentially abundant taxa from faecal content (ln change and p-value < 0.05) among repeated samplings at family (a) and genus (b) level. Only predominant (> 1%) significant families and genera are presented; positive values and negative values indicate greater and lower abundance, respectively, in repeated samplings (d7, d14, d21 and weaned piglet) compared to new-born piglets (d2); taxa are sorted according to the general mean of relative abundance (the average of the entire trial, indicated between brackets in %) and in decreasing order. Figure created with the online open-source tool Datawrapper (http://​dataw​rapper.​de) Saladrigas‑García et al. Animal Microbiome (2022) 4:68 Page 9 of 23 Fig. 2 (See legend on previous page.) Saladrigas‑García et al. Animal Microbiome (2022) 4:68 Page 10 of 23 Fig. 3 Bar diagram of the relative abundances (%) of the main (> 1%) families (a) and genera (b), organized by farm (Alpha or Bravo) and by age. WP = Weaned piglets Saladrigas‑García et al. Animal Microbiome (2022) 4:68 Page 11 of 23 (p = 0.002), Rikenellaceae RC9 gut group (p = 0.002) and and piglet age. In accordance with the richness results, Parabacteroides (p = 0.036). Exceptionally, the Bacteroides greater alpha diversity was observed in 21-day-old pig- genus was found to be more enriched in the piglets from lets compared to day 2, as presented in Additional file 1: the Alpha farm (p = 0.019). Table S4. Moreover, Delta and Foxtrot farms were found to have significantly lower alpha-diversities for Observed Trial 2: impact of the farm management practices species, Chao1 and Shannon indices than Charlie and on piglets’ microbiota Echo (p = 0.033, p = 0.034, and p = 0.027, respectively Changes in microbiota structure and biodiversity for farm effect) despite at day 2 of life decreases in alpha- An average of 74,429 ± 3,611 amplicon sequence variants diversity were only found in Foxtrot farm (Fig. 5). No sig- (ASV) were obtained per sample for a total of 102 sam- nificant impact of the use of medicated diet was found. ples of faecal content. As found in the previous trial, a Regarding beta diversity calculated by means of the greater richness was observed in 21-day-old piglets when Whittaker index, no differences were found, neither compared to 2-day-old piglets (p < 0.001). Among the dif- between the sampling days, nor among farms or the use of ferent farms, no difference was detected in terms of rich- medicated feed in sows. However, the multivariate analy- ness, although a higher number of reads was observed in sis of ecological communities by the ANOSIM Bray– the Foxtrot farm (p = 0.057). Curtis dissimilarities matrix, showed a significant effect Regarding changes in the microbial ecosystem with of farm (p = 0.011 and p = 0.001, for d2 and d21, respec- age, farm environmental factors and the use of medicated tively) and of use of medicated feed in sows (p = 0.011 feed in the sows, PERMANOVA multivariate analysis and p = 0.001, for d2 and d21, respectively). In 2-day-old showed that all factors considered (age, farm and medi- piglets, a greater similarity was observed among the sam- cated feed) had a significant impact on shaping the gut ples from the Foxtrot farm, while the rest of the farms communities (p < 0.001 for age factor and p = 0.001 for presented similar results among them. However, on the farm and feed). However, the non-metric multidimen- 21st day of life, it was the Charlie farm that stood out for sional scaling (NMDS) based on the Bray–Curtis dis- its greater similarity among samples. Regarding the use of tance, only found significant the effect of age (p < 0.001) medicated feed in mothers, in 2-day-old piglets, a greater with two clear clusters between 2-day-old piglets and similarity was observed among the microbiota of the pig- 21-day-old piglets (Fig. 4). lets from medicated mothers (ABF), while on day 21 the The diversity indices also revealed important dif- piglets from the non-medicated groups (NMF) showed a ferences among farms, use of medicated feed in sows, greater similarity among each other. Fig. 4 NMDS of the relative abundances of ASV in Trial 2. Different colours have been used to highlight each sampling day and different shapes to distinguish each farm. A significant impact shaping the gut communities is produced by the piglets’ age (p < 0.001) with two clear clusters between 2-day-old piglets and 21-day-old piglets. A greater richness was observed in 21-day-old piglets when compared to 2-day-old piglets (p < 0.001). Farm and medicated feed were not found to be significant by the NMDS analysis Saladrigas‑García et al. Animal Microbiome (2022) 4:68 Page 12 of 23 Fig. 5 Box plot of the alpha diversity calculated using the Chao1 (a), Shannon (b) and Simpson indices (c) of the four farms included in Trial 2 in 2-day-old piglets. The Foxtrot farm showed lower alpha diversity values than the rest of the farms, suggesting a possible effect of maternal medicated feed Changes in taxonomic groups significantly lower levels of Lachnospiraceae and Fuso- Proteobacteria and Firmicutes constituted the two pre- bacteriaceae and higher levels of Enterococcaceae dominant phyla in the faecal microbiota of 2-day-old were observed in the Foxtrot farm (Table 4). At genus piglets, contributing with a 58.0% and 30.6% of the rela- level UCG-002, Desulfovibrio and Butyricimonas also tive abundance, respectively. Bacteroidetes (7.1%) and showed different abundance among farms (p < 0.001; Fusobacteria (2.8%), with a representation greater than p = 0.016 and p = 0.001, respectively) (Fig. 7). 1% in relative abundance, were also considered pre- Regarding the possible impact of the use of medicated dominant phyla. At day 21 of life, a very different sce- feed in the microbiota of the progeny, Fig. 8 shows those nario was observed, with Firmicutes and Bacteroidetes taxonomic groups (families and genera) that were sig- being the two predominant phyla in the faecal micro- nificantly modified at days 2 and 21 of life. At day 2 of biota of the piglet, contributing 49.19 and 35.73% of the life, piglets from dams fed non-medicated feed (NMF) relative abundance, respectively. Proteobacteria (9.73%) showed statistically significant lower abundances of Fir- and Fusobacteria (1.14%), were also considered pre- micutes and Fusobacteria phyla explained by the lower dominant phyla. Changes at genus level with the age of detected levels of Fusobacteriaceae, Prevotellaceae, Sut- the pig were also remarkable and are shown in Fig. 6. terellaceae, Enterococcaceae and Acidaminococcaceae From a total of 476 genera detected only 27 taxa repre- families. Regarding genera, Fusobacterium, Prevotella, sented a relative abundance greater than 1%. Phascolarctobacterium, Sutterella and Enterococcus also Regarding the possible effects of the farm on micro- showed significant lower abundances. Moreover, NMF bial taxonomy, Tables 3 and 4 show the relative abun- farms were associated with higher counts of the Entero- dances of the predominant families (> 0.5%), in the bacteriaceae family and the Escherichia/Shigella genera. different farms, on days 2 and 21 respectively, and Fig. 7 On day 21 of life, changes were only observed in minor shows differences in the genus level. Two days after taxonomic groups (Fig. 8). birth, significantly higher levels for the Bacteroidaceae family were detected at the Echo farm. Moraxellaceae family was the lowest in the Foxtrot and the highest in Discussion the Delta farm. Trends were detected for other families, The main findings of this study include a detailed with remarkably higher levels (p = 0.059) of Enterobac- description of the gut microbial colonization pattern in teriaceae in the Foxtrot farm (Table 3). At the genus the commercial piglet and a first analysis of the potential level, Sutterella, Desulfovibrio and Blautia also showed impact of the farm husbandry guides on such process. different abundance among farms (p = 0.025; p = 0.035 Most studies on pig microbiota have been conducted and p = 0.006 respectively) (Fig. 7). At day 21 of life, under standardized experimental conditions, and from Saladrigas‑García et al. Animal Microbiome (2022) 4:68 Page 13 of 23 Fig. 6 Bar diagram of the relative abundances (%) of the main genera (> 1%) in Trial 2, organized by farm (Charlie, Delta, Echo or Foxtrot) and by sampling day. In Echo and Foxtrot farms the sows received medicated feed this point of view, this is probably one of the few stud- welfare [34]. Therefore, understanding the dynamics of ies performed in real farms where the situation differs the young piglet gut microbiota during the first weeks of largely. In the following sections, we will discuss the pos- life is certainly concerning as its a major impact on the sible definition of the gut microbial colonisation process future health and growth performance of pigs. in the piglet and those management or environmental Several authors have affirmed that age and weaning are variables capable to condition such pattern. the driving factors of microbiota development, point- ing out specific changes in specific taxonomic groups at certain ages [16, 20–22, 32, 35, 36]. Initially, the gut colo- The pattern of microbial colonization during the first nization of newborn piglets is characterized by bacteria weeks of life belonging to the Clostridiaceace, Enterobacteriaceae, After birth, the intestinal microbiome of the piglet rap- Fusobacteriaceae, and Bacteroidaceae families, which are idly undergoes a remarkable shift from the initial micro- progressively replaced by carbohydrate fermenting bacte- bial groups which are present during the first days of ria, essentially the acetate, propionate, and butyrate-pro- life to the establishment of an adult-like microbial com- ducing microorganisms. During the weaning transition, munity, experiencing in between a period of changing the microbial ecosystem evolves from a microbiome microbial successions [12, 32, 33]. This initial pattern oriented to the degradation of milk carbohydrates, com- of microbial maturation is essential as the gut micro- posed of families like Bacteroidaceae and Lactobacil- biota is fundamental for the adequate development and laceae, towards a more intricate one. programming of the mucosal immune response [13]. Finally, microorganisms belonging to the Lachno- Moreover, a “window of opportunity” has been described spiraceae and Ruminococcaceae families, adapted to during this early-life development stage, where any dis- metabolize a wide range of complex oligosaccharides ruption may have long-lasting impacts on health and Saladrigas‑García et al. Animal Microbiome (2022) 4:68 Page 14 of 23 Table 3 Relative abundances (%) of the families present in the faecal microbiota of 2-day-old piglets from Trial 2 in a percentage higher than 0.5%, classified by farm and in decreasing order of abundance in relation to the general average Charlie Delta Echo Foxtrot SEM P-value Enterobacteriaceae 37.38 35.76 44.72 71.82 4.393 0.059 Clostridiaceae 26.33 25.55 14.93 17.03 2.699 0.999 Alcaligenaceae 7.31 7.84 2.78 0.59 1.728 0.999 a a b a Bacteroidaceae 3.13 1.50 11.19 2.33 0.996 0.031 Streptococcaceae 3.33 4.44 3.45 1.54 0.605 0.079 Lachnospiraceae 1.73 4.58 4.16 1.42 0.620 0.183 Fusobacteriaceae 5.88 2.94 0.64 0.25 0.996 0.653 Pasteurellaceae 0.99 2.22 3.18 2.37 0.480 0.359 Burkholderiaceae 0.23 1.00 4.18 0.48 0.859 0.647 ab a ab b Moraxellaceae 1.20 3.03 1.04 0.12 0.655 0.030 a no_match 0.98 1.91 0.62 0.20 0.304 0.506 Veillonellaceae 0.54 1.20 1.75 0.17 0.236 0.094 Peptostreptococcaceae 0.63 0.39 1.48 0.17 0.175 0.094 Prevotellaceae 1.36 0.74 0.18 0.04 0.221 0.889 Suerellaceae 1.48 0.32 0.11 0.20 0.184 0.295 Lactobacillaceae 0.69 0.54 0.42 0.21 0.079 0.629 Oscillospiraceae 0.48 0.63 0.37 0.04 0.151 0.135 Enterococcaceae 0.34 0.77 0.13 0.09 0.063 0.219 Muribaculaceae 0.26 0.25 0.02 0.01 0.068 0.999 a no_match = Not assigned taxa. ■: Firmicutes ■: Bacteroidetes ■: Fusobacteria ■: Proteobacteria □: None The colours represent the phyla to which the families belong. In Echo and Foxtrot farms, sows received medicated feed; in Charlie, Delta and Echo farms piglets were offered a rehydrating solution during lactation. Significant changes are highlighted in bold. and polysaccharides while producing SCFA, have been weaning [37, 44, 45], with a later increase from weaning reported to begin to emerge after weaning [17, 37]. to adulthood. This discrepancy might be due to differ- For instance, the gradual increase observed in the ences among studies in weaning age or the post-weaning microbial diversity of piglets as they aged, in the present sampling time-point but also to differences in manage- study, is in accordance with several previous works [16, ment and feeding practices or in the microbiological 17, 21, 22, 32, 36, 38–40]. A higher diversity in the gut environment of the farm. Regarding variability of the microbiota has been related to a more mature gut ecosys- microbiota among individual piglets (interindividual tem and is in agreement with the concept of functional Bray–Curtis distances), several authors have described redundancy, which supports that additional taxa add that it tends to diminish as the piglets age [17, 22, 40, 46], redundancy to specific functions, helping the ecosystem suggesting that gut microbiota structure of piglets tends to preserve its resilience and stability after environmen- to converge among animals as they age to establish of a tal stress [22, 41–43]. Diversity results are, however, con- homogenous, rich and stable microbiota composition tradictory with some other studies that have reported a after weaning. However, results from the present work decreased alpha diversity during the early period after showed that this evolution is not always present, as in Trial 1 beta diversity was increased as the piglets grew. It Saladrigas‑García et al. Animal Microbiome (2022) 4:68 Page 15 of 23 Table 4 Relative abundances (%) of the families present in the faecal microbiota of 21-day-old piglets from Trial 2 in a percentage higher than 0.5%, classified by farm and in decreasing order of abundance in relation to the general average Charlie Delta Echo Foxtrot SEM P-value Bacteroidaceae 9.37 13.95 10.18 19.05 1.459 0.252 a a a b Lachnospiraceae 10.99 14.12 12.75 8.31 0.677 0.043 Oscillospiraceae 14.50 8.62 9.34 10.92 0.600 0.901 Prevotellaceae 8.31 8.37 11.95 7.48 0.979 0.407 Enterobacteriaceae 3.76 5.48 5.33 10.26 1.155 0.107 no_matcha 6.40 6.50 6.07 4.57 0.484 0.219 Muribaculaceae 8.06 5.52 5.60 3.43 0.697 0.270 Ruminococcaceae 4.47 4.28 6.93 5.26 0.421 0.747 Rikenellaceae 5.36 4.31 3.79 1.97 0.367 0.051 Lactobacillaceae 2.56 5.34 4.00 2.03 0.397 0.107 Christensenellaceae 4.53 4.01 2.09 2.98 0.316 0.382 Acidaminococcaceae 2.44 2.14 1.92 2.77 0.131 0.329 Erysipelotrichaceae 1.38 2.06 1.89 2.42 0.245 0.562 Tannerellaceae 0.75 0.72 1.68 2.34 0.176 0.782 Marinifilaceae 2.01 0.73 1.44 1.04 0.173 0.500 Desulfovibrionaceae 1.63 0.90 1.40 1.11 0.085 0.365 p-2534-18B5_gut_group 1.96 1.24 0.79 0.69 0.225 0.747 Clostridiaceae 0.94 1.18 0.40 1.69 0.264 0.181 a a a b Fusobacteriaceae 1.19 1.30 1.54 0.05 0.321 0.005 Spirochaetaceae 0.94 0.84 0.89 1.26 0.132 0.999 a a a b Enterococcaceae 0.32 0.64 0.34 1.83 0.286 0.005 UCG-010 0.83 0.82 0.67 0.40 0.064 0.155 Comamonadaceae 0.26 0.05 0.66 1.64 0.320 0.433 Campylobacteraceae 0.61 0.28 1.24 0.42 0.156 0.400 Anaerovoracaceae 0.45 0.86 0.67 0.49 0.073 0.999 Methanobacteriaceae 0.60 0.54 0.49 0.36 0.050 0.175 Veillonellaceae 0.34 0.47 0.68 0.38 0.075 0.490 Erysipelatoclostridiaceae 0.36 0.31 0.58 0.37 0.068 0.719 F082 0.12 0.06 0.06 0.13 0.031 0.182 a no_match = Not assigned taxa. ■: Firmicutes ■: Bacteroidetes ■: Fusobacteria ■: Proteobacteria ■: Spirochaetes ■: Euryarchaeota □: None The colours represent the phyla to which the families belong. In Echo and Foxtrot farms, sows received medicated feed; in Charlie, Delta and Echo farms piglets were offered a rehydrating solution during lactation. Significant changes are highlighted in bold. Saladrigas‑García et al. Animal Microbiome (2022) 4:68 Page 16 of 23 Fig. 7 Differentially abundant taxa from faecal content (ln change and p-value < 0.05) among farms at genus level in 2-day-old (a) and 21-day-old (b) piglets. Only predominant (> 0.5%) significant genera are presented; positive values and negative values indicate greater and lower abundance, respectively, among farm environments (Delta, Echo and Foxtrot) compared to Charlie farm; taxa are sorted according to the general mean of relative abundance (the average of the entire trial, indicated between brackets in %) and in decreasing order. Figure created with the online open-source tool Datawrapper (http://​dataw​rapper.​de) (See figure on next page.) Fig. 8 Ln changes in taxa promoted by sow feed (sows receiving (ABF) vs not (NMF) medicated feed; ln change and p-value < 0.05) at phylum, family and genus level in the microbiota of piglets. Positive values and negative values indicate higher and lower abundance, respectively, in piglets from ABF-fed mothers. Taxa are sorted by level of significance (from higher to lower). Only taxa with relative abundances higher than 0.5% are included in the figure. The presented differences are based only on taxa detected in at least half of the animals per sampling. Figure created with the online open-source tool Datawrapper (http://​dataw​rapper.​de) is possible that under the complex and challenging envi- design and conditions, pig genetics and environmen- ronmental conditions the animals have to face in com- tal factors including the time of the year the sampling mercial practice, the evolution of their gut ecosystems was performed, the sampling procedures or the analyti- will diverge more widely than when they are reared under cal procedures. A trend towards higher abundance was highly standardized experimental conditions. observed for the phyla Spirochaetes as the piglets aged Firmicutes followed by Bacteroidetes were found to be in both Trials, as observed by Pajarillo et al. [32]. At the the dominant phyla across all experimental samplings family level, relative abundances of Enterobacteriaceae, except for 2-day-old piglets. Although this is consistent Clostridiaceae, Fusobacteriaceae, and Veillonellaceae with the majority of earlier studies [16, 19, 20, 22, 32, 36, declined over time while those of Prevotellaceae, Rumi- 39], there are a few reports with Bacteroidetes or Proteo- nococcaceae, Spirochaetaceae, Rikenellaceae, Erysipel- bacteria [21, 47] as the pre-dominant phyla. However, otrichaceae and Succinovibrionaceae increased. Similar the age of the piglet plays an important role in this result. results have also been reported in previous studies [20, In the present study, we observed that in 2-day-old pig- 32, 33, 46]. However, contrary to what was reported by lets from Trial 1 Proteobacteria was found to be almost these authors, we did not observe a decrease in the Bac- as abundant as Firmicutes and in Trial 2, Proteobacte- teroidaceae family in Trial 1 or an increase in the Veil- ria doubled Firmicutes in relative abundance. There- lonellaceae family. Bacteroidaceae, Lactobacillaceae, fore, the Proteobacteria phylum stands out in this study Lachnospiraceae and Streptococcaceae families showed for its important weight at the time closest to birth. The oscillations in their relative abundances throughout the decreasing abundance of Fusobacteria observed in Trial repeated samplings, generally increasing during lactation 1 during the first weeks of life has also been reported by and slightly decreasing after weaning. several other authors [17, 21, 32, 38–40]. However, some The present study pinpointed the early intestinal colo- studies have not reported the presence of this bacterial nizers belonging to Bacteroides, Escherichia-Shigella, group at all [16, 35]. This is in consonance with the results Clostridium sensu stricto 1 and Fusobacterium genera. observed in Trial 2, where much lower abundances than This is in accordance with Petri et al. [48], who reported those previously reported in Trial 1 were detected. Such the genera Escherichia, Clostridium, Fusobacterium, differences in taxonomic abundance could, to some Streptococcus, and Enterococcus to be the earliest colo- extent, be due to variability factors such as the study nizers of the pig gut, between birth and 2 days. However, Saladrigas‑García et al. Animal Microbiome (2022) 4:68 Page 17 of 23 Fig. 8 (See legend on previous page.) Saladrigas‑García et al. Animal Microbiome (2022) 4:68 Page 18 of 23 a high level of individuality has been reported to occur was relatively controlled since all the selected farms were in 1- and 2-week-old piglets, indicating that there is indoors, close-cycle, geographically located in the same considerable randomness to the process of acquiring region of Spain, and vertically integrated using the same microbes [8]. Although this gut community in very young breed and feed formula. Moreover, farms included in piglets might be highly dynamic, the microbial com- Trial 1 or Trial 2 were sampled in the same temporal munity is known to stabilize by day 28. Decreases in the period. Differences among farms were mostly due to the abundances of Clostridium, Fusobacterium and Escher- metaphylactic use of antimicrobials (injected after birth ichia-Shigella with the age of the piglets have also been to piglets and/or given to the sows as medicate feed) observed by several other authors [16, 17, 19, 22, 32, 46]. and rehydrating solutions during lactation. Farms were During lactation, the genera Bacteroides and Lactobacil- also selected based on differences in historical records lus acquire greater relative importance with respect to of digestive (B. hyodysenteriae) and respiratory (Actino- the rest of the genera and this fact has been correlated bacillus pleuropneumonia, APP) diseases (see Table 1 with a milk-oriented microbiome [16]. Bacteroides has for additional information). After Trial 1, days 2 and 21 been reported to use a wide range of both milk oligosac- were chosen as the most relevant sampling days in Trial charides and host-derived glycans [49] and Lactobacil- 2 to characterize the evolution pattern of the gut micro- lus is a well-known lactate producer from lactose [50]. bial ecosystem after birth and to assess possible factors Moreover, whereas Fusobacterium has been positively influencing this process. Day 2 would reflect one of the correlated to intestinal diseases [51–53], Lactobacillus moments of greater variability in the establishment pro- has been labelled as a major player in the establishment cess of the first colonizers and day 21 would give a rela- and maintenance of bacterial homeostasis after birth [2]. tively accurate picture of the first ecological equilibrium The second significant change in the microbial com- reached by the gut microbiota after birth. munity of the piglet occurs in the period around wean- As expected, significant differences among farms were ing. In the present study, the increase in genera such as found in the microbial ecosystem of piglets in both tri- Prevotella, Butyricimonas, Christensenellaceae R-7 group, als. In Trial 1, greater alpha diversity was found in Dorea, Phascolarctobacterium, Rikenellaceae RC9 gut the Bravo farm, whereas in Trial 2, lower alpha diversity group, Subdoligranulum and Ruminococcaceae UCG-002 was observed in the Foxtrot farm in 2-day-old piglets. stands out. This is largely in agreement with previous This difference among farms was also demonstrated by observations which exemplify Prevotella as a prominent the fact that several taxa were influenced by the rear- actor in the typical post-weaning microbiota together ing farm. Although the impact of the effect of the rear- with species belonging to Roseburia, Faecalibacterium, ing farm on specific taxa has not been analysed in detail Ruminococcus, Lachnospira, Dorea, Blautia, Subdol- in the literature, changes in the microbiota of the piglets igranulum [16, 17, 19–21, 32, 33, 35, 37, 40, 46, 54]. The have been reported [46, 57]. Indeed, in addition to animal drastic increase in the relative abundance of Prevotella age and genetic background [58], the structure and activ- is likely due to the established capacity of the members ity of gut microbiota can differ among animals depending of this genus to metabolize plant-derived non-starch on various factors including dietary influence [16, 59, 60], polysaccharides to short-chain fatty acids (SCFAs) [55, sanitary status [61], antimicrobial use [14, 62–64] and 56], which are prominent constituents of the cereal- husbandry practices [65, 66], among others. For instance, based weaner diet. Therefore, the abrupt change to a in Trial 1, piglets from Alpha farm received an intra- solid cereal-based diet and the withdrawal of milk would muscular dose of amoxicillin (15 mg amoxicillin/kg BW) explain the decrease of the previously mentioned genera between their second and fifth day of life, which could and the increase of propionate- and butyrate-producing explain the lower alpha diversity found in Alpha piglets. genera. Altogether, the higher abundance of propionate- Although most of the existing literature has focused on and butyrate-producing genera in older piglets, adapted the study of oral administration of antibiotics, a recent to digest resistant starches and dietary fibres, show the study has evidenced significant dysbiosis regardless of quick microbial transformation of the piglets’ gut micro- administration route in mice [67]. Moreover, amoxicil- biota to cope with diets rich in complex carbohydrates. lin has been related to profound dysbiotic effects, with population richness and diversity significantly reduced, The impact of farm management and environment on gut in orally treated pigs [62]. In Trial 1 we also observed microbial colonization of piglets that 2 and 7-days-old piglets from the Alpha farm pre- In the present study, 6 commercial farms were included sented lower abundances of pathogenic bacteria such in order to determine the extent to which variations as Clostridium sensu stricto 1 and Escherichia-Shigella, in the farm environment and rearing conditions influence whereas Lactobacillus counts were increased in Bravo the microbiota development after birth. Farm variability piglets in most of the time-points (d2, d7, d21 and after Saladrigas‑García et al. Animal Microbiome (2022) 4:68 Page 19 of 23 weaning). Similar results have been reported in orally administration during late gestation has been reported treated pigs with amoxicillin, where the antibiotic inter- to impact both sow vaginal and faecal microbial diversi- vention decreased the abundance of Lactobacillus and ties [69, 70]. Together, these results would suggest that other lactic acid bacteria [14, 62]. However, all these dif- maternal antibiotic treatment might influence the gut ferences could had been due not only to the use of amoxi- microbial colonization of the offspring through changing cillin but also to differences in the composition of the maternal microbiota composition. In addition, it is well oral solutions used in both farms that in Alpha not only known that maternal antibiotic residues can be trans- included electrolytes but also a blend of mono, di and tri- ferred from mothers to their offspring via breastfeeding glycerides of medium chain fatty acids, together a blend [71]. To date, very few studies have analysed the effect of acidifiers. With time and increasing age, the piglets of the administration of antibiotics to mothers on their from the Bravo farm ended up showing a more mature offspring, and there are especially few studies focused on microbiota after weaning, with greater abundances of the effects produced in their intestinal microbiota. Arnal butyrogenic genera such as Prevotella, Coprococcus, Fae- et al. [70] reported a significant effect on the microbiota calibacterium and Dorea, among many others, as well of the small intestine of the offspring but not of the colon. as lower abundances of Escherichia-Shigella and Fuso- Moreover, some effects of maternal antibiotic treatment bacterium that could have been due to a lower selective on the gut physiology and morphology of the offspring pressure from antimicrobial prophylaxis. However, dif- have been seen in early-life [69, 70, 72, 73]. In Trial 2 only ferences between farms after weaning in Trial 1 should farms Echo and Foxtrot gave the sows medicated feed be regarded with caution as the Alpha farm was sampled during lactation. Two-day-old piglets from dams treated earlier than Bravo (7 vs. 14 days post-weaning) due to an with medicated feed showed lower abundances of pro- imminent metaphylactic treatment of the piglets after pionate- and butyrate-producing genera, such as Phas- the first signs of diarrhoea outbreak. This could explain colarctobacterium and Prevotella, respectively. Lower the higher abundance of Escherichia-Shigella and Fuso- abundances of Enterococcus were also observed. Entero- bacterium in Alpha piglets and the slower development coccus genera have recently gained attention due to their of the fermentative bacteria. ability to produce bacteriocins recognized for their wide- In Trial 2, differences were also observed among rear- range effectiveness on pathogenic and spoilage bacteria ing farms. In addition to the recurring B. hyodysenteriae [74]. This is in consonance with the higher abundances problems in Echo and Foxtrot farms, which implied the of Escherichia-Shigella observed in piglets from dams consumption of medicated feed by the mothers, the main treated with medicated feed. Piglets from treated dams management difference consisted of the absence of the also showed lower abundances of Fusobacterium at day rehydrating solution in the piglets from the Foxtrot farm. 2 and 21 of life. Some authors have suggested that the Interestingly, in Foxtrot farm a decreased alpha diversity abundance of Fusobacteria, is positively correlated with at day 2 and a marked increased abundance of Entero- diarrheal swine diseases, such as the porcine epidemic bacteriaceae both at 2 and 21 days of life were observed. diarrhoea and the new neonatal porcine diarrhoea [51, Moreover, at 21 days of life, significant changes in other 52]. On day 21 of life, lower abundances of the acetate- microbial groups were also observed in this farm, such as and butyrate-producing genera Rikenellaceae RC9 gut a greater abundance of Enterococcaceae and a lower rela- group and CAG-873, respectively, were also observed in tive abundance of Lachnospiraceae and Fusobacteriaceae. the piglets of mothers fed with medicated feed. Higher Although it is not possible to assure that the observed abundances of Rikenellaceae RC9 gut group are sugges- changes are due to either the initial antibiotic treatment tive of a high level of functional redundancy for acetate or the re-hydrating solutions supplied during the first in the swine gut microbiome [75]. A greater abundance week of life of the piglets, it is evidenced that differential of Parabacteroides was also observed in piglets from farm management guidelines, and particularly antimicro- medicated dams. Higher abundances of Parabacteroides bial prophylaxis, can affect the development of the intes- have been linked with lower BW and ADG [76, 77]. tinal microbiota of the pig early in life. These findings suggest that maternal antibiotic treat- Regarding the use of antibiotics in the mothers, results ment affected the gut microbiota of offspring through the from Trial 2 showed ecological changes in the micro- transfer of maternal gut microbiota to newborn piglets, bial communities of the piglet associated with this fac- although it is difficult to specify whether the maternal tor. The impact of maternal antibiotic treatment on the antibiotic treatment affected the microbiota development progeny has been a subject of study especially in the of offspring in a direct or indirect process. Although field of human medicine. It has been recently found that some researchers have stated that maternal antibiotic antibiotic use during pregnancy alters the commensal treatment and early antibiotic administration affect the vaginal microbiota of women [68]. Moreover, amoxicillin development of intestinal microbiota of the piglets, along Saladrigas‑García et al. Animal Microbiome (2022) 4:68 Page 20 of 23 with piglet mucosal tissue gene expression [11, 62, 63, 69, diverse early colonization. A greater evenness between 78–81], further research is needed concerning the conse- minor bacterial groups could be considered as beneficial quences of maternal antibiotic treatment with regards to since it could represent an increased capacity for adap- the gut microbiome of their offspring. tation and resilience. Actually, in Charlie, Delta, Echo, Comparing both Trials (1 and 2), the process of the gut and Foxtrot farms a very high abundance of Escherichia- microbial colonization of piglets followed a similar evo- Shigella is observed (around 40%, although reaching up lution pattern between different farms, both in terms of to 71% on the Foxtrot farm) whereas, in Alpha and Bravo species richness and microbiota diversity, which gradu- farms, the Escherichia-Shigella, Bacteroides, and Fuso- ally increased in piglets with age, and in relation to the bacterium genera share similar abundances (around 10%, taxonomic groups involved in this process, despite the without any of them standing out excessively). However, high individual variability observed at the earlier stages. despite these results, Fusobacterium has been typically However, in line with the high variability expected associated with diarrhoea and gut inflammation [52, at this early age [8], there is great variability among the 82, 83]. Moreover, it should be noted that within Trial 2 six farms noticeable even from the phylum level (Addi- farms, Echo and Foxtrot showed particularly low relative tional file 1: Table S5). Thus, in Alpha and Bravo farms abundances of Fusobacterium, being those the farms in there are large initial abundances of Fusobacteria, while which antibiotics were administered to the sows. Actu- this difference seems to be occupied by Proteobacteria ally, Fusobacterium has been reported to reduce signifi- in the rest of the farms. Similarly, the phylum Firmicutes cantly after an antibiotic treatment [52]. In any case, all even doubles its relative abundance in the Alpha and these data would point out Fusobacterium as a possible Delta farms in comparison with the Foxtrot farm and microbial community marker that would deserve more drastic changes are also observed in the Bacteroidetes attention in future studies. group. Similar results are observed at the family level, There are very few works, that have tried to assess the with large differences among farms and piglets of the impact of the environment or rearing practices on the same age. While in Charlie, Delta, Echo, and Foxtrot the establishment of the intestinal microbiota of the piglets predominant family is Enterobacteriaceae, in Alpha and after birth in commercial conditions. Recently, Lüh- Bravo the Fusobacteriaceae family also plays an impor- rmann et al. [84] analyzing the faecal microbiota from tant role, becoming the predominant family in the case of sows and their piglets, at two time points each, in 20 the Alpha farm. Concerning Clostridia, Clostridicaeae is German farms (sows ante- and postpartum and piglets observed in all farms with an average relative abundance before and after weaning) were also able to identify dif- of around 20%, with quite a similarity between different ferences related to farms, particularly when they analyze farms. Therefore, although it is true that the predominant the family-unit mother-piglet, suggesting the existence of groups in two-day-old piglets are Enterobacteriaceae and a farm-specific microbiome of sows and piglets. Despite Clostridiaceae, there is some controversy with the Fuso- these encouraging results, authors also recognize the dif- bacteriaceae family. The high abundance of Fusobacteria ficulty of making a comprehensive analysis of the role observed in Alpha and Bravo farms (Trial 1) during the that feeding, housing, and management could have in first days of life has also been reported by several other such definition. Undoubtedly, more studies are needed to studies [21, 32, 38–40]. However, some studies have not fully elucidate the factors that could condition such farm- reported the presence of this bacterial group at all [16, specific microbiomes and their role in the health and per- 35]. Such differences in taxonomic abundance could, to formance of piglets. some extent, be due to uncontrolled environmental dif- ferences between Trials (each trial was run in a different Conclusions time window) but also to methodological bias. Despite all Taken together, the present study confirms and refines samplings being carried out using the same procedures the knowledge about microbiome development during and samples analyzed in the same lab with the same early-life stages in piglets. The initial colonization char- methodology, certain batch effect cannot be discarded. acterized by bacteria belonging to the Clostridiaceace, Regarding the distinctive higher abundance of Fuso- Enterobacteriaceae and Fusobacteriaceae families is pro- bacteria in Alpha and Bravo farms (Additional file 1: gressively replaced by fermenting bacteria, essentially Table S6) it is interesting to highlight that higher abun- the acetate, propionate and butyrate-producing microor- dances of Fusobacteria were associated with a greater ganisms. Moreover, this study evidences that the rearing abundance of minority genera representing less than 1% farm and particularly the use of antimicrobials in the sow of the total (all together close to 30% of relative abun- can have a significative impact on this process. Feeding dance vs. 8.9% in farms form Trial 2). These results would antibiotics to the mothers can induce ecological changes suggest Fusobacterium as a possible indicator of a more in the piglets without changes in microbiota richness or Saladrigas‑García et al. Animal Microbiome (2022) 4:68 Page 21 of 23 diversity. Significant shifts can be observed in particular University of Barcelona. Faecal swab collections were performed as per stand‑ ard operative procedures approved by the Animal Ethics Committee. microbial groups, and between them, lower abundances of Fusobacterium in those piglets from medicated sows. Consent for publication This work also evidences that the use of oral rehydrat- Not applicable. ing solutions can modulate the piglets’ microbiota with Competing interests significant reductions in the abundance of the Enterobac- The authors declare no competing interests. The funders had no role in the teriaceae family. All in all, our results demonstrate that design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. the farm and the rearing practices, particularly the use of antimicrobials in the mothers, can determine struc- Author details tural and taxonomic changes in the gut microbiota of the 1 Servicio de Nutrición Y Bienestar Animal. Departamento de Ciencia Animal Y de los Alimentos, Universitat Autònoma de Barcelona, 08193 Bellaterra, young piglet. More studies will be needed to fully under- Barcelona, Spain. 2 Grupo Vall Companys, 25191 Lleida, Spain. stand the underlying phenomena before designing inter- vention strategies addressed to improve the resilience of Received: 26 January 2022 Accepted: 18 December 2022 the piglet intestinal ecosystem. Abbreviations References ANOSIM: Analysis of similarities; ANOVA: Analysis of variance; APP: Actinobacil‑ 1. Houghteling P, Walker W. 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PLoS One. 2015;10:e0118092. https://​doi.​org/​10.​1371/​journ​al.​ • thorough peer review by experienced researchers in your field pone.​01180​92. • rapid publication on acceptance 73. Trevisi P, Luise D, Correa F, Bosi P. Timely Control of Gastrointestinal Eubio‑ • support for research data, including large and complex data types sis: A Strategic Pillar of Pig Health. Microorganisms. 2021;9. 74. Hanchi H, Mottawea W, Sebei K, Hammami R. The genus enterococcus: • gold Open Access which fosters wider collaboration and increased citations between probiotic potential and safety concerns—an update. Front • maximum visibility for your research: over 100M website views per year Microbiol. 2018;9:1791. 75. Holman DB, Brunelle BW, Trachsel J, Allen HK. Meta-analysis to define a At BMC, research is always in progress. core microbiota in the swine gut. mSystems. 2017;2:1–14. Learn more biomedcentral.com/submissions