TYPE Original Research PUBLISHED 05 December 2022 DOI 10.3389/fmicb.2022.982712 Influence of dietary n-3 OPEN ACCESS long-chain fatty acids on EDITED BY Zhipeng Li, Jilin Agriculture University, China microbial diversity and REVIEWED BY Shiyu Tao, composition of sows’ feces, Huazhong Agricultural University, China Zhiyong Fan, colostrum, milk, and suckling Hunan Agricultural University, China *CORRESPONDENCE piglets’ feces Núria Tous nuria.tous@irta.cat Eudald Llauradó-Calero1 , Eric Climent2 , Empar Chenoll2 , SPECIALTY SECTION This article was submitted to Maria Ballester3 , Ignacio Badiola4 , Rosil Lizardo1 , Microorganisms in Vertebrate Digestive Systems, David Torrallardona1 , Enric Esteve-Garcia1 and Núria Tous1* a section of the journal 1 Animal Nutrition, Institute for Food and Agricultural Research and Technology (IRTA), Tarragona, Frontiers in Microbiology Spain, 2 ADM Biopolis, Paterna, Spain, 3 Animal Breeding and Genetics, Institute for Food and RECEIVED 30 June 2022 Agricultural Research and Technology (IRTA), Tarragona, Spain, 4 Animal Health-CReSA, Institute for ACCEPTED 07 November 2022 Food and Agricultural Research and Technology (IRTA), Bellaterra, Spain PUBLISHED 05 December 2022 CITATION Llauradó-Calero E, Climent E, Chenoll E, Ballester M, Badiola I, Introduction: Very little is known about the impact of n-3 long-chain fatty Lizardo R, Torrallardona D, Esteve-Garcia E and Tous N (2022) acids (n-3 LCFAs) on the microbiota of sows and their piglets. The aim of this Influence of dietary n-3 long-chain study was to evaluate the effect of n-3 LCFA in sow diets on the microbiota fatty acids on microbial diversity and composition of sows’ feces, composition of sows’ feces, colostrum, and milk as well as that of piglets’ colostrum, milk, and suckling piglets’ feces. feces. Front. Microbiol. 13:982712. Methods: Twenty-two sows were randomly assigned to either a control or doi: 10.3389/fmicb.2022.982712 an n-3 LCFA diet from service to weaning. Sows’ and piglets’ performance COPYRIGHT was monitored. The gestating and lactating sows’ microbiomes in feces, © 2022 Llauradó-Calero, Climent, Chenoll, Ballester, Badiola, Lizardo, colostrum, and milk were characterized by 16s ribosomal RNA gene Torrallardona, Esteve-Garcia and Tous. sequencing. The fecal microbiome from the two lowest (>800 g) and the This is an open-access article distributed under the terms of the two highest birth weight piglets per litter was also characterized, and the LPS Creative Commons Attribution License levels in plasma were analyzed at weaning. (CC BY). The use, distribution or reproduction in other forums is Results and Discussion: n-3 LCFA increased microbiota alpha diversity in permitted, provided the original author(s) and the copyright owner(s) suckling piglets’ and gestating sows’ feces. However, no effects were observed are credited and that the original in colostrum, milk, or lactating sows’ feces. Dietary n-3 LCFA modified the publication in this journal is cited, in accordance with accepted academic microbiota composition of gestating sows’ feces, milk, and suckling piglets’ practice. No use, distribution or feces, without affecting lactating sows’ feces or colostrum. In gestating reproduction is permitted which does sows’ feces and milk, the decrease in genus Succinivibrio and the increase not comply with these terms. of Proteobacteria phylum, due to the increased genera Brenneria and Escherichia, respectively, stand out. In the feces of suckling piglets, the higher abundance of the beneficial genus Akkermansia and Bacteroides, and different species of Lactobacillus are highlighted. In addition, positive correlations for families and genera were found between lactating sows’ feces and milk, milk and suckling piglets’ feces, and lactating sows’ feces and suckling piglets’ feces. To conclude, dietary n-3 LCFA had a positive impact on the microbiome of suckling piglet’s feces by increasing microbial diversity and some beneficial Frontiers in Microbiology 01 frontiersin.org Llauradó-Calero et al. 10.3389/fmicb.2022.982712 bacteria populations, had a few minor modifications on the microbiome of milk and gestating sows’ feces and did not change the microbiome in lactating sows’ feces or colostrum. Therefore, this study shows the effect of dietary n-3 LCFA on the microbiota of sows, colostrum, milk, and suckling piglets during the lactation period providing crucial information on the microbiota status at the early stages of life, which have an impact on the post-weaning. KEYWORDS gestating and lactating sows, suckling piglets, n-3 long-chain fatty acids, microbial communities, colostrum, milk, microbial transference Introduction present reduced growth rates (Lopez-Verge et al., 2018) and they are less likely to survive. Nutritional strategies applied to The microbiota of pigs is composed of hundreds of different the sow may become an effective tool to shape the microbiota microorganisms and their acquisition and establishment are establishment of piglets to improve their growth and immune influenced by different external factors. Concretely, the development. predominant immediate postnatal factors that determine Although under commercial conditions the ingredients in initial microbial definition in newborn piglets are likely sow diets contain considerable amounts of n-6 long-chain fatty colostrum, milk, feed, oral-fecal transmission, and the neonatal acids (LCFAs) and the fat sources used are rich in saturated fatty environment (Nowland et al., 2019; Lauridsen, 2020). However, acids, over the last few years the study of the inclusion of n-3 the exact time of initial colonization in pigs has not yet been LCFA sources in sow diets has gained interest (Tanghe and De determined, and some previous reports described prenatal Smet, 2013). Concretely, research on n-3 LCFA in sow nutrition microbial colonization driven by the mother, so it is reasonable has mainly focused on their influence on milk composition and to assume that sows’ microbiota would also significantly offspring performance (Lauridsen, 2020; Llauradó-Calero et al., influence the microbiota of their offspring before or during 2021). Moreover, due to their anti-inflammatory effects (Calder, parturition (Nowland et al., 2021). Moreover, the establishment 2010) and capability to influence the epithelial barrier functions of the gut microbiota has become a key factor in piglet survival, (Liu, 2015), the impact of n-3 LCFA on immune status (Huber as suggested by previous studies in humans showing that et al., 2018) and intestinal epithelium function (Liu et al., 2012) an appropriate intestinal microbiota resulting from optimal has also been evaluated. However, little is known about the colonization may improve health and limit diseases (Nowland influence of n-3 LCFA on microbiota diversity and composition et al., 2019). After all, the microbiota is an important regulator in sows’ feces, colostrum, milk, and piglets’ feces. of mammal physiology and could exert several beneficial roles Considering the sow as the first and most relevant factor in digestion, protection against pathogens, maintenance of for the establishment of an optimal microbiota in the newborn normal function of intestinal villi, and regulation of the immune piglet, the aim of the current study was to evaluate the inclusion response (Gresse et al., 2017). In addition, optimal colonization of n-3 LCFA, concretely a solid fish oil rich in eicosapentaenoic during lactation could become a critical factor considering acid (EPA) (C20:5 n-3) and docosahexaenoic acid (DHA) (C22:6 that the change from maternal milk to solid food during n-3), in sow diets and their impact on the microbiota of gestating weaning entails microbial modifications that coincide with the and lactating sows’ feces, colostrum, milk and the feces of morphological and functional maturation of the gut barrier, and suckling piglets. The impact of n-3 LCFA on the sow to piglet with important changes in the gut immune system (Hooper, microbial transference was also analyzed. 2004; Beaumont et al., 2021). Genetic selection for hyperprolific sows has resulted in a substantial increase in litter size, and this increase in the Materials and methods sows’ prolificacy has resulted in an increased proportion of piglets born with a low weight (< 1.0 kg birth weight) (Quiniou Ethics statement et al., 2002). Low birth weight piglets present a greater energy requirement per kg of body weight (Noblet and Etienne, 1987), Institute for Food and Agricultural Research and and a poorer thermoregulatory ability, which is combined with Technology’s (IRTA) Ethical Committee on Animal a limited colostrum intake (Edwards and Baxter, 2015) due to Experimentation approved the use of animals for this being less vigorous when competing for the limited number experiment in accordance with Directive 2010/63/EU of of teats in hyperprolific sows. As consequence, these animals 22 September 2010 and according to the recommendation of Frontiers in Microbiology 02 frontiersin.org Llauradó-Calero et al. 10.3389/fmicb.2022.982712 the European Commission 2007/526/CE, the Spanish guidelines FEDNA specifications (de Blas et al., 2013), and their ingredient for the care and use of animals in research (B.O.E. number and nutrient composition were already described by Llauradó- 34, Real Decreto 53/2013) and the regional regulations on the Calero et al. (2021). Diets were formulated to contain the same use and handling of experimental animals (Decree 214/97, level of nutrients (metabolizable energy, crude protein, digestive Generalitat de Catalunya) (project number: 10294). lysine, and ether extract) except for the fat content. In control diets, dietary fat was included using a common animal fat source (5 Sysfeed ; Sysfeed SLU, Granollers, Spain) at 15 and 30 g/kg in R Animals, experimental design, and the gestation and lactation phases, respectively. In the test diets housing (n-3 LCFA), 15 g/kg of fat was replaced (totally during gestation and one-half during lactation) by solid fish oil (Lipomega ; V&S R Twenty-two sows in two batches (12 and 10 sows met the Asociados, Madrid, Spain). Creep feed diets for piglets contained selection criteria of being between the third and the sixth parity, 30 g/kg of animal fat or an equivalent amount of solid fish oil in respectively) were fed one of the two experimental diets from the control or n-3 LCFA diet, respectively. service until the end of lactation (c.a. 28 days post-farrowing). Feed intake of sows was restricted to a maximum of 3 kg/day Within each batch, sows were grouped regarding their body during the gestation period and progressively increased after weight and their parity number into pairs, as similar as possible, farrowing until reaching ad libitum feed intake. Piglets’ creep and sows in each pair were randomly assigned to either a control feed was offered ad libitum from day 11 post-farrowing. or an n-3 long-chain fatty acid (n-3 LCFA) diet. At birth, the two piglets with the lowest (>800 g) (LBW) and the two piglets with the heaviest (HBW) body weight in each litter were selected Growth measurements and sampling for future microbiome analyses. Cross-fostering of piglets was performed only during the first 24 h of life to standardize litter Sows were weighed at service, 1 week before farrowing size to 12 piglets per sow, whenever possible, solely among sows (when moved to the farrowing crates), the day after farrowing, belonging to the same experimental treatment, and without and at weaning. In addition, backfat thickness in the P2 involving selected piglets. Sow feeds were provided ad libitum in position of sows was also measured at the same time points self-dispensing feeding hoppers. At 11 days of age, piglets were (except the day after farrowing) through ultrasound scanning offered either a control or an n-3 LCFA creep feed according (Piglog 105 ; Frontmatec, Kolding, Denmark). Daily feed intake R to the corresponding maternal diet, using floor-attached round was monitored and recorded individually during gestation feeders. Water was provided ad libitum from nipple drinkers. and lactation. The average daily gain of gestation period was Sows were allocated in individual stalls from service until calculated from service to day 107 of gestation (1 week before pregnancy confirmation. Once confirmed, they were group- farrowing), and the average daily gain of lactation period housed in a gestation barn until 1 week before farrowing. Sows from 1 day after farrowing to weaning (c.a. 28 days post- were then relocated to individual farrowing crates (0.7 x 2 farrowing). At birth, the total number of piglets born, piglets m) in pens equipped with partially slatted floors and a heated born alive/dead, mummies, and their individual weights were floor panel for piglets (set at 32–34◦ C). The farrowing room recorded for each sow. During lactation, the weight of piglets was lit with natural light from a window and with fluorescent was monitored at 24 h, at 20 days of age, and weaning. artificial light (manually operated), and ventilated via single, Cross-fostering was performed within 24 h after birth and variable-speed fans linked to temperature sensors. The inside the 24 h recordings were considered as the initial values for temperature of the building was automatically controlled, and litter characteristics and growth performance of suckling piglets the target temperature of the stables at farrowing was set during lactation. In the same way, the average daily gain of at 24◦ C and it was reduced by 0.5◦ C per week during the litters and piglets was calculated from 24 h post-farrowing to lactation period. each weighing time. Creep feed disappearance was monitored Unfortunately, one sow from the second batch (n-3 from day 11 of lactation until weaning. One sow from the LCFA treatment) farrowed out of the scheduled time, without n-3 LCFA group gave birth to less than six piglets and the supervision, and it was not possible to record the litter corresponding data for litter characteristics at birth (obtained characteristics at birth and sample colostrum, so the sow was before performing cross-fostering) were excluded from the removed from the trial. analysis. Individual animals were selected for the microbiome studies. Fecal samples from all the sows were collected 1 week before Experimental diets parturition (day 107 of gestation) and at weaning after removing the piglets (c.a. day 28 post-farrowing). At birth, the two lowest Barley–corn-based gestation and lactation diets for sows and (> 800 g) and the two highest birth-weight piglets in each litter the creep feed for piglets were formulated in accordance with were selected and their feces were sampled at weaning. In all Frontiers in Microbiology 03 frontiersin.org Llauradó-Calero et al. 10.3389/fmicb.2022.982712 cases, samples were individually preserved with Real stock buffer OTUs at 99.7% of identity. The BLAST tool was used to (Durviz, Paterna, Spain) and stored at −80◦ C until analysis. taxonomically identify each OTU against the National Center Colostrum samples from each sow were obtained for Biotechnology Information (NCBI) 16S rRNA database immediately after the birth of the first piglet and milk (20th December 2020) using BLASTn version 2.10.0+. samples were collected at weaning after the piglet’s removal. Sows were milked from all mammary glands following i.v. injection of 1.0 ml of oxytocin (20 IU/ml) (Super’s Diana S.L., Lipopolysaccharides measurement in Parets del Vallès, Spain). The samples from the different nipples plasma of suckling piglets in each sow were pooled and aliquots of at least 3 ml were immediately frozen and stored at −80◦ C until analysis for The sandwich ELISA kit Porcine Lipopolysaccharides microbiome determination. (LPSs) ELISA Kit (MBS269464; MyBioSource, San Diego, Blood samples from selected piglets were collected at CA, USA) was employed according to the manufacturer’s weaning. Blood was obtained by jugular venipuncture in instructions for the quantitative measurement of LPS tubes with ethylenediaminetetraacetic acid (EDTA) and was in all plasma samples from weaned piglets. Intra-assay kept at 4◦ C a maximum of 120 min until centrifugation precision and inter-assay precision of the kit were ≤ 8% (3,000 rpm, 10 min). Plasma aliquots for LPS measurement and ≤ 12%, respectively. were obtained and stored at −80◦ C for a maximum of 30 min after centrifugation. Statistical analysis Deoxyribonucleic acid extraction and The analysis of variance of growth and performance data bacterial 16S gene amplification and of gestating and lactating sows and suckling piglets and sequencing LPS concentration data of plasma from suckling piglets were performed through the GLIMMIX procedure of SAS software For microbiome analysis, DNA from feces was isolated (SAS/STAT 14.1; SAS Institute Inc., Cary, NC, USA). For growth with the aid of QIAamp Power Fecal Pro DNA Kit (Qiagen, and performance measurements, dietary treatment was included Hilden, Germany), with bead beating and enzymatic lysis in the model as the fixed effect and batch as the random effect. steps prior to extraction to avoid bias in DNA purification Sow body weight at the beginning of the trial and the parity toward the misrepresentation of gram-positive bacteria. For number were initially introduced into the model as covariates. milk and colostrum, samples were processed with bead beating However, only the initial sow body weight was included in and enzymatic lysis steps followed by the Blood & Tissue the statistical analysis since parity had no significant effect. In Kit (Qiagen) and further DNA concentration. To evaluate addition, the variable days of lactation were included in the the bacterial composition, massive genome sequencing of the model as a covariate for all the data at weaning. In terms of hypervariable region V3–V4 of the bacterial 16s rRNA gene was stillborn piglets, deaths, and mummies, data were normalized √ conducted. Samples were amplified using key-tagged eubacterial using a square root transformation ( X+0.5), but the means of primers (Klindworth et al., 2013) and sequenced on a MiSeq the original data are presented in tables. For LPS concentrations, Illumina Platform, using a 2 × 300nt paired-end strategy, the model included dietary treatment as the fixed effect and sow following Illumina Library preparation and sequencing for as the random effect. All data were tested using Kolmogorov– metagenomic studies protocol (Zhang et al., 2014; Marcel, 2011). Smirnov test to identify possible outliers and values were excluded if P < 0.01. Results were expressed as means ± SD. Analysis of sequencing data Significant differences were set at P < 0.05. The resulting sequences were split considering the barcode Microbiome analyses were done using R software (R Core introduced during the PCR reaction. PEAR program version Team, 2012). Alpha and beta diversity indexes were obtained 0.9.1 was used to overlap R1 and R2 reads (overlap of 50 nt using the vegan package, as implemented for R version 3.2.3 and quality of the overlap with a minimum of Q20 (Zhang (Oksanen et al., 2020). Bray–Curtis distances were selected to et al., 2014), providing a single FASTQ file for each of the analyze beta diversity, and their significance was studied with samples. Cutadapt v2.6 (Marcel, 2011) was used to trim 16S PERMANOVA tests. DESeq2 package from R (Love et al., rRNA PCR primers and sequences were treated with a quality 2014) was used to generate a generalized linear model with filter, removing low-quality fragments (under Q20 in Phred fixed effects (control vs. n-3 LCFA diet) with negative binomial scale) and short sequences (under 200nt). Chimeric sequences family to compare operational taxonomic unit (OTU) counts that potentially arise during the PCR amplification step were between groups and select the potential bacterial biomarkers. identified de novo using CD-HIT software v4.8.1 (Li et al., P-values were corrected for multiple testing with Benjamini 2012) and removed. CD-HIT software was also used to create and Hochberg method, and the statistical significance cutoff Frontiers in Microbiology 04 frontiersin.org Llauradó-Calero et al. 10.3389/fmicb.2022.982712 was set at P < 0.05. The ratios Firmicutes/Bacteroidetes and genera, and species levels. The largest differences in terms of Lactobacillus/Proteobacteria were calculated through relative alpha diversity were observed in the feces of suckling piglets. An abundances and adjusted to a maximum value of 100. Higher increase in microbiota diversity calculated through Simpson and values were considered outliers and were removed from the Shannon indices in all studied levels was detected in piglets from analysis. Significant differences were set at P < 0.05, while the n-3 LCFA sows compared with piglets from the control sows. tendencies at P < 0.1 using Mann–Whitney–Wilcoxon test. In addition, microbiota diversity was significantly increased by The PROC CORR procedure of SAS software was used dietary fish oil through the Simpson index in feces from n-3 to obtain the Pearson correlation coefficient of the relative LCFA gestating sows. abundances of differential microbial populations regardless Alpha-diversity in terms of Richness, Simpson, and of the dietary treatment between gestating sows’ feces and Shannon indices in colostrum, lactating sow’s feces, or milk were colostrum, lactating sows’ feces and milk, milk and sucking not significantly affected by fish oil in the sows’ diet (Table 1). piglets’ feces, and lactating sows’ feces and suckling piglets’ feces to study the sow-piglet microbial transference and between suckling piglets’ feces and growth measurements of suckling Microbiota composition and piglets to identify possible microbial populations affecting the differential microbial communities animal growth. A significant correlation level was set at r > 0.5 and P < 0.05. The PCoA analysis of sows and piglets’ feces, colostrum, and milk regardless of experimental dietary treatments is presented in Figure 1. PERMANOVA revealed distinct clusters for each Results sample type (R2 = 0.32; P = 0.001). Beta-dispersion analysis showed differences among all the different types of samples Sows’ weight, feed Intake, litter analyzed (all P ≤ 0.037) except between feces of gestating and characteristics, and piglets’ weight lactating sows (P = 0.235). The fecal microbiome of gestating sows was composed The body weight, backfat thickness, and feed intake of sows mainly of phylum Firmicutes, Families Clostridiaceae, during gestation and lactation are presented in Supplementary Peptostreptococcaceae, Lactobacillaceae, Streptococcaceae, Table 1. Sow’s body weight at service (P = 0.494), 107 days of Erysipelotrichaceae, and Lachnospiraceae, and genera gestation (P = 0.207), one day after farrowing (P = 0.336), and at Clostridium, Lactobacillus, Streptococcus, Terrisporobacter, weaning (P = 0.812), and sow’s average daily weight gain during and Turicibacter (Figure 2). Lower abundances of the family gestation (P = 0.202) and lactation (P = 0.278) did not differ Succinivibrionaceae (P = 0.020) and genus Succinivibrio between dietary treatments. In the same way, backfat thickness (P = 0.031) were observed in the feces of sows from the n-3 in the P2 position at service (P = 0.696), 107 days of gestation LCFA group than those from the control group (Figure 3). (P = 0.795), and at weaning (P = 0.502), and the average daily The core microbiome obtained in colostrum samples feed intake during gestation (P = 0.787) or lactation (P = 0.683) was represented by phyla Firmicutes, Actinobacteria, did also not differ either between treatments. and Proteobacteria. Staphylococcaceae, Lactobacillaceae, The number of piglets born, piglets born alive/dead, Clostridiaceae, and Moraxellaceae were the dominant mummies, piglet’s weight, average daily gain during lactation, Families and Staphylococcus, Lactobacillus, Clostridium, and creep feed intake are presented in Supplementary Table 2. and Streptococcus represented the most abundant genera Litter characteristics and the average piglet’s body weight did (Figure 2). No differential bacterial communities were detected not show any significant differences between treatments at birth, between control and n-3 LCFA treatments at any taxonomical 24 h after birth, 20 days after birth, or at weaning (all P ≥ 0.166). level studied (Figure 3). In the same way, no effects of dietary fish oil were observed In the feces of lactating sows, Firmicutes was also the for litter or piglet average daily weight gain during the first 20 most abundant phylum. Families Peptostreptococcaceae, days (P = 0.339 and P = 0.553, respectively) or during the whole Clostridiaceae, Erysipelotrichaceae, Lactobacillaceae, and lactation period (P = 0.974 and P = 0.714, respectively). Piglet Lachnospiraceae and genera Clostridium, Terrisporobacter, creep feed disappearance did not differ between control and n-3 Turicibacter, and Lactobacillus had the highest abundance LCFA diets (P = 0.599) either. (Figure 2). As for colostrum, no significant differences between the control and n-3 LCFA groups were observed for any taxa at any taxonomical level (Figure 3). Microbiota diversity In milk, Firmicutes, Proteobacteria, and Actinobacteria were the dominant bacterial phyla followed by Bacteroidetes. Table 1 shows richness and diversity values for each At the family and genus level, the dominant taxa were experimental group and type of sample at phylum, family, Lactobacillaceae, Streptococcaceae, Peptostreptococcaceae, Frontiers in Microbiology 05 frontiersin.org Llauradó-Calero et al. 10.3389/fmicb.2022.982712 TABLE 1 Microbiota alpha diversity indices, calculated at phylum, family, genera, and species level, of feces of gestating and lactating sows, colostrum, milk, and feces of suckling piglets from animals fed either control or n-3 LCFA diets.1 Alpha diversity indices Richness Simpson index Shannon index Taxonomy level Control n-3 LCFA P-value Control n-3 LCFA P-value Control n-3 LCFA P-value Gestating sows’ feces Phylum 7.65 ± 0.63 7.83 ± 0.55 0.48 0.57 ± 0.02 0.55 ± 0.02 0.11 1.01 ± 0.08 0.96 ± 0.09 0.21 Family 27.0 ± 1.28 27.1 ± 1.33 0.83 0.74 ± 0.04 0.77 ± 0.04 0.073 1.90 ± 0.10 1.94 ± 0.15 0.47 Genera 50.1 ± 1.44 49.6 ± 2.59 0.55 0.74 ± 0.04 0.77 ± 0.04 0.068 2.08 ± 0.11 2.12 ± 0.19 0.49 Species 80.1 ± 3.14 81.3 ± 4.96 0.52 0.75 ± 0.04 0.79 ± 0.05 0.037 2.31 ± 0.14 2.45 ± 0.25 0.14 Colostrum Phylum 8.04 ± 0.78 8.19 ± 0.35 0.58 0.54 ± 0.14 0.54 ± 0.08 0.93 1.08 ± 0.24 1.08 ± 0.14 0.94 Family 52.1 ± 4.98 52.8 ± 3.36 0.69 0.82 ± 0.17 0.83 ± 0.09 0.83 2.50 ± 0.53 2.47 ± 0.41 0.91 Genera 102 ± 19.5 102 ± 9.93 0.92 0.84 ± 0.16 0.85 ± 0.10 0.78 2.81 ± 0.60 2.81 ± 0.49 0.99 Species 206 ± 43.8 200 ± 29.4 0.72 0.91 ± 0.07 0.91 ± 0.09 0.99 3.58 ± 0.54 3.58 ± 0.56 0.97 Lactating sows’ feces Phylum 8.18 ± 0.40 8.06 ± 0.35 0.46 0.59 ± 0.04 0.54 ± 0.07 0.064 1.06 ± 0.10 0.97 ± 0.15 0.091 Family 21.9 ± 0.76 21.9 ± 1.16 0.97 0.75 ± 0.04 0.77 ± 0.03 0.51 1.85 ± 0.13 1.84 ± 0.13 0.80 Genera 48.0 ± 1.77 47.6 ± 2.42 0.61 0.77 ± 0.05 0.78 ± 0.04 0.42 2.10 ± 0.19 2.10 ± 0.13 0.96 Species 76. 4 ± 2.86 77.9 ± 4.23 0.36 0.78 ± 0.05 0.80 ± 0.05 0.34 2.36 ± 0.23 2.38 ± 0.14 0.77 Milk Phylum 8.00 ± < 0.01 7.82 ± 0.60 0.33 0.56 ± 0.09 0.58 ± 0.11 0.58 1.12 ± 0.17 1.17 ± 0.21 0.57 Family 45.6 ± 3.45 46.6 ± 1.81 0.49 0.86 ± 0.05 0.88 ± 0.04 0.34 2.49 ± 0.16 2.62 ± 0.21 0.11 Genera 97.1 ± 7.81 97.1 ± 7.88 0.99 0.87 ± 0.05 0.89 ± 0.04 0.36 2.80 ± 0.20 2.93 ± 0.24 0.16 Species 208 ± 23.6 207 ± 25.0 0.92 0.90 ± 0.06 0.92 ± 0.04 0.34 3.43 ± 0.29 3.63 ± 0.30 0.14 Suckling piglet’s feces Phylum 9.83 ± 0.89 9.50 ± 0.74 0.064 0.68 ± 0.06 0.71 ± 0.07 0.036 1.36 ± 0.18 1.46 ± 0.21 0.026 Family 35.8 ± 2.76 35.3 ± 2.77 0.45 0.78 ± 0.07 0.83 ± 0.07 0.006 2.09 ± 0.21 2.28 ± 0.29 0.001 Genera 72.7 ± 5.87 70.0 ± 7.18 0.065 0.81 ± 0.07 0.84 ± 0.06 0.021 2.45 ± 0.23 2.56 ± 0.25 0.054 Species 118 ± 10.5 117 ± 12.9 0.62 0.82 ± 0.07 0.86 ± 0.07 0.009 2.69 ± 0.28 2.89 ± 0.34 0.005 LCFA, long-chain fatty acid. 1 Values are means ± SD of 11 control and 10 n-3 LCFA samples from sows (gestating and lactating sows’ feces, colostrum, and milk) or 44 control and 40 n-3 LCFA samples from piglets (suckling piglets’ feces). Staphylococcaceae, and Lachnospiraceae, and Lactobacillus, Bacteroidaceae, Lactobacillaceae, Ruminococcaceae, and Streptococcus, Staphylococcus, and Clostridium, respectively Clostridiaceae were the most abundant families, and Bacteroides, (Figure 2). Differences between the control and the n-3 LCFA Lactobacillus, Gemmiger, Clostridium, and Methanobrevibacter treatments were observed for specific groups (Figure 3). the most abundant genera (Figure 2). Piglet feces were the Concretely, Phylum Proteobacteria (P = 0.017) was increased by type of sample that presented a larger number of differences dietary n-3 LCFA due to the increased families Pectobacteriaceae in microbial communities between treatments. Concretely, (P = 0.020) and Enterobacteriaceae (P = 0.020) and the main one phylum, 15 families, and 27 genera differed between genera belonging to these families, Brenneria (P = 0.026) treatments (Figure 3). Phylum Verrucomicrobia (P = 0.045) and Escherichia (P = 0.026), respectively. In addition, other and genera Akkermansia (P = 0.041) were increased in the changes by the inclusion of dietary fish oil were detected feces of piglets from n-3 LCFA-fed sows. In addition, and such as decreases in the family Ruminococcaceae (P = 0.023) within Firmicutes, dietary n-3 LCFA reduced the family and genera Ruminococcus (P = 0.026), increases in family Ruminococcaceae (P < 0.001) due to reductions in the genera Propionibacteriaceae (P = 0.020) and genera Cutibacterium Gemmiger (P = 0.032) and Ruminococcus (P = 0.036). Within (P = 0.026), and decreases in family Spirochaetaceae (P = 0.023) the same phylum, the family Clostridiaceae (P = 0.002) and and genera Treponema (P = 0.028). genera Clostridium (P = 0.001) were increased by dietary Microbiota composition in the feces of suckling piglets was n-3 LCFA. Family Enterococcaceae (P = 0.010) and genera dominated by phyla Firmicutes, Bacteroidetes, Proteobacteria, Enterococcus (P = 0.011) were also increased by fish oil. It Euryarchaeota, and Actinobacteria. Lachnospiraceae, should also be pointed out that the feces from piglets in Frontiers in Microbiology 06 frontiersin.org Llauradó-Calero et al. 10.3389/fmicb.2022.982712 FIGURE 1 Principal coordinates analysis (PCoA) plot of gestating and lactating sows’ feces (n = 21, respectively), colostrum (n = 21), milk (n = 21), and suckling piglets’ feces (n = 84), regardless of experimental dietary treatments, revealed distinct clusters for each sample type (R2 = 0.32; P = 0.001). PERMANOVA test performed using Bray–Curtis distances to analyze beta-dispersion showed differences between all the different types of samples analyzed (all P ≤ 0.037) except among gestating and lactating sows’ feces (P = 0.235). the n-3 LCFA group had a reduced abundance of genus genera Synergistes (P = 0.002) but not modifying phylum Holdemanella (P = 0.032), Flintibacter (P = 0.024), and Synergistetes or family Synergistaceae. Anaerotaenia (P = 0.009), and an increased abundance of Finally, the ratios Firmicutes/Bacteroidetes and genus Streptococcus (P = 0.023) and Erysipelatoclostridium Lactobacillus/Proteobacteria were also compared between (P < 0.001). Regarding phylum Bacteroidetes, dietary fish treatments for all types of samples, and this is reported in oil increased in piglets’ feces in the families Tannerellaceae Figure 4. In the feces of lactating sows, an increase in the ratio (P < 0.001) and Odoribacteraceae (P = 0.042) due to the Firmicutes/Bacteroidetes (P = 0.040) and a tendency to decrease increase in their respective genera Parabacteroides (P < 0.001) the ratio Lactobacillus/Proteobacteria were observed (P = 0.072) and Butyricimonas (P = 0.002). Within the same phylum also with the n-3 LCFA inclusion, while a tendency to reduce the stands out the increased abundance of genera Bacteroides ratio Firmicutes/Bacteroidetes (P = 0.070) was observed in feces (P = 0.009) by n-3 LCFA. In terms of phylum Proteobacteria, of suckling piglets of n-3 LCFA group. For feces of gestating an increase of families Pectobacteriaceae (P = 0.047) and sows, colostrum, and milk, no differences between treatments were observed for either, Firmicutes/Bacteroidetes ratio (all Enterobacteriaceae (P = 0.023), due to the increase in their P ≥ 0.22) or Lactobacillus/Proteobacteria ratio (all P ≥ 0.19). respective genera Brenneria (P < 0.001) and Escherichia (P < 0.001), and a reduction of family Desulfovibrionaceae (P = 0.006) were observed in piglets from sows fed the n-3 LCFA. In the phylum of Actinobacteria, n-3 LCFA reduced The concentration of family Coriobacteriaceae (P < 0.001) and genera Collinsella lipopolysaccharides in the plasma of (P < 0.001), and increased family Actinomycetaceae (P < 0.001) suckling piglets and genera Schaalia (P = 0.007), and family Atopobiaceae (P < 0.001), including genus Olsenella (P < 0.001) and As shown in Figure 5, no differences in plasma LPS Atopobium (P < 0.001). In addition, n-3 LCFA also reduced concentration were observed between samples from piglets of Frontiers in Microbiology 07 frontiersin.org Llauradó-Calero et al. 10.3389/fmicb.2022.982712 FIGURE 2 Microbiota composition by the relative abundance of the top 10 phyla (A), top 20 families (B), and top 20 genera (C) of feces of gestating and lactating sows (n = 21), colostrum (n = 21), milk (n = 21), and feces of suckling piglets (n = 84) according to dietary treatment (control vs. n-3 LCFA). LCFA, long-chain fatty acids. Frontiers in Microbiology 08 frontiersin.org Llauradó-Calero et al. 10.3389/fmicb.2022.982712 and Ruminococcus in feces with Anaerostipes, Ruminococcus, and Treponema in milk (Figure 6). Between milk and feces of suckling piglets, two positive correlations were observed at the family level and five positive correlations at the genus level (Figure 6). Concretely, at the family level, Odoribacteraceae and Tannerellaceae in milk were positively correlated with Tannerellaceae in piglets’ feces. At the genus level, Akkermansia, Bacteroides, Flintibacter, and Parabacteroides in milk were also positively correlated with Parabacteroides in feces and Erysipelatoclostridium in milk with Olsenella in feces. One positive correlation at family and genus levels was observed between feces from lactating sows and feces from suckling piglets (Figure 6). Specifically, the correlations were between the family Atopobiaceae and genus Atopobium in lactating sows’ feces with the family Peptoniphilaceae and genus Akkermansia in suckling piglets’ feces, respectively. Finally, no significant correlations between microbial populations were observed at any taxonomical level between gestating sow’s feces and colostrum, and between bacterial populations in suckling piglets’ feces and the growth measurements of suckling piglets during lactation. Discussion The microbiota plays an integral role in influencing host metabolism, its immune system, and the development of a healthy gastrointestinal tract. In addition, the health of offspring is strongly linked to microbial exposure throughout life (Nowland et al., 2019). Considering that weaning is a critical FIGURE 3 part of pig life characterized by being a complex and stressful Heatmap representing the differentially abundant phyla, families, event due to the dietary, social and environmental changes and genera between control and n-3 LCFA dietary treatments in (Gresse et al., 2017), the acquisition of an optimal microbiota feces of gestating and lactating sows, colostrum, milk, and feces of suckling piglets. For feces of gestating and lactating sows, during the suckling period becomes crucial. Few studies on mice colostrum, and milk; n = 11 for control and n = 10 for n-3 LCFA. and humans have studied the effect of n-3 fatty acids on the gut For feces of suckling piglets; n = 44 for control and n = 40 for n-3 LCFA. Significant differences between treatments were set microbiota; however, it is described as a poorly understood topic at P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***). LCFA, (Costantini et al., 2017). To our knowledge, no prior studies have long-chain fatty acids. evaluated the impact of n-3 LCFA on the microbial diversity and composition of sows’ feces, colostrum, and milk and the feces of their suckling piglets, and therefore research on the impact of the control group and piglets of the n-3 LCFA group (181 ± 98.4 n-3 LCFA on swine microbiota deserves further attention. and 162 ± 77.8 pg/ml, respectively; P = 0.700). The effects of the inclusion of n-3 LCFA in sows’ diets on sow’s weight, litter characteristics, and growth performance of piglets during lactation have been previously reported (Tanghe Correlations of differential microbial and De Smet, 2013). The animals selected for the current study populations between sample types are part of a larger trial studying the impact of n-3 LCFA inclusion in the diets of gestating and lactating diets of sows on The comparison of feces from lactating sows and their different parameters including performance (Llauradó-Calero milk rendered positive correlations at the family level, et al., 2021), and a trend to increase average piglet body weight Ruminococcaceae in feces with Ruminococcaceae and at weaning is reported, which was in the line with the results of Spirochaetaceae in milk, and four positive correlations at Rooke et al. (2001). However, with the group of sows selected the genus level, Anaerostipes in feces with Anaerostipes in milk, for the current study, no differences in performance between Frontiers in Microbiology 09 frontiersin.org Llauradó-Calero et al. 10.3389/fmicb.2022.982712 FIGURE 4 Firmicutes/Bacteroidetes (A) and Lactobacillus/Proteobacteria (B) ratios calculated between treatments in all sample types. For feces of gestating and lactating sows, colostrum, and milk; n = 11 for control and n = 10 for n-3 LCFA. For feces of suckling piglets; n = 44 for control and n = 40 for n-3 LCFA. Significant differences between treatments were set at P < 0.05 (*), while tendencies were set at P < 0.10 (†). Ratios were adjusted at a maximum value of 100 and higher values were removed by being considered outliers. LCFA, long-chain fatty acids. FIGURE 5 Lipopolysaccharide (LPS) concentration in plasma of suckling piglets from control and n-3 LCFA-fed sows. n = 44 for control and n = 40 for n-3 LCFA. LCFA, long-chain fatty acids. treatments were observed, which would also be consistent with microbiota (Simpson et al., 2000; Niu et al., 2019). However, the results of Lauridsen and Danielsen (2004) and Leonard et al. in gestating sows, microbial diversity increased when they were (2010). fed with the n-3 LCFA diet. In addition, the decrease of family Despite diet being one of the main factors affecting intestinal Succinivibrionaceae due to the decrease of genus Succinivibrio microbiota (Duan et al., 2019), in the current study, the stands out, which is considered a core microbiome of the inclusion of fish oil as a source of n-3 LCFA in sow diets cause proximal colon or cecum of swine and is related to propionate minor changes in microbial populations in the feces of the formation and decarboxylation process (Bergamaschi et al., sows. This may be due to the fact that sows, as adult animals, 2020). However, in lactating sows, no differences in diversity are characterized by presenting a stable and well-developed or populations at family and genus levels were found between Frontiers in Microbiology 10 frontiersin.org Llauradó-Calero et al. 10.3389/fmicb.2022.982712 FIGURE 6 Correlations between modified microbial families (A) and genera (B) from feces of lactating sows (navy blue, n = 21), milk (light blue, n = 21), and feces of suckling piglets (purple, n = 84). Pearson correlation coefficient (r) in green indicates a positive correlation. A significant correlation level was set at r > 0.5 and P < 0.05. control and n-3 LCFA-fed animals. Even so, in lactating of families Pectobacteriaceae and Enterobacteriaceae and the sows, an increase in the ratio of Firmicutes/Bacteroidetes was increase in genera Brenneria and Escherichia, respectively. observed for the n-3 LCFA group. More Firmicutes and fewer While no effects of Brenneria have been described on animal Bacteroidetes have been suggested as a characteristic of fat pigs microbiota, Escherichia is a common inhabitant in swine gut and have been related to fat deposition (Zhao et al., 2015), microbiota although some of its species could be pathogenic although we found no differences between treatments for the (Schierack et al., 2007). In addition, the decrease of the family weight of the sows or their backfat thickness on P2 position at Ruminococcaceae due to the decrease of the genus Ruminococcus weaning. stands out. Ruminococcaceae has been reported to play a role Both colostrum and milk play a critical role in piglets’ in the degradation of complex carbohydrates (Crost et al., development since they are the first sources of nutrients 2018) and the production of butyrate, and it is associated for newborn piglets which are characterized as having low with anti-inflammatory effects (Liu et al., 2019). Particularly, energy reserves and being immunologically naive. In addition, Ruminococcus OTU was identified as R. flavefaciens, which colostrum and milk not only provide nutrients, energy, plays an important role in the digestion of hemicellulose and and immunity, but they also enable the establishment of cellulose plant cell walls (Fontes and Gilbert, 2010), and their commensal microbes (Nowland et al., 2019). Given that degradation-derived products may act as prebiotics to gut optimal colostrum and milk supply are crucial for intestinal microbiota (Rajan et al., 2021). microbiota colonization and development (Nowland et al., Compared to sows, suckling piglets experienced larger 2021), the impact of n-3 LCFA on colostrum and milk modifications in the diversity and bacterial populations of their microbiota composition becomes very relevant. In the current fecal microbiota due to n-3 LCFA. However, we cannot be study, the bacterial compositions of colostrum and milk were sure whether the higher diversity in the fecal samples from shown to be different, regardless of dietary treatment. On n-3 LCFA piglets is due to the direct effect of dietary n-3 the one hand, the microbiota of colostrum was dominated LCFA in creep feed, vertical transmission from the sow at mainly by phyla Firmicutes, Actinobacteria, and Proteobacteria birth, or a combination of both. In addition, no effects of and families Staphylococcaceae, Lactobacillaceae, Clostridiaceae, birth weight category (low vs. high birth weight piglets) or and Moraxellaceae. On the other hand, the microbiota of interactions between dietary treatment and piglets’ birth weight milk was dominated by phyla Firmicutes, Proteobacteria, were observed for either diversity or bacterial populations. and Actinobacteria and Lactobacillaceae, Streptococcaceae, This contrasts with the results reported by Li et al. (2018), Peptostreptococcaceae, Staphylococcaceae, and Lachnospiraceae Li N. et al. (2019) who described differences between LBW and at the family level. Interestingly, dietary n-3 LCFA impacted normal birth weight piglets during the suckling period for the differently on the microbial populations from colostrum and microbiota of feces and the microbiota of digesta in the ileum milk. In colostrum, no changes were observed in microbial and colon. Regarding dietary treatment, bacterial alpha diversity diversity nor differential populations due to n-3 LCFA according to Simpson and Shannon indices was increased in supplementation. In milk, however, one phylum, six families, suckling piglets on the n-3 LCFA diet. In a previous study, and seven genera were modified by the n-3 LCFA diet, although Djuric et al. (2019) described an increase of colonic bacterial no changes in microbial diversity were observed. Among these diversity in healthy human adults following dietary fish oil changes, the increase in Proteobacteria should be highlighted supplementation which they related to the anti-inflammatory since it is one of the most dominant phyla in milk microbiota. effects of n-3 LCFA, agreeing with Calder (2019), who suggested This increase was mainly due to the higher abundance a new mechanism by which n-3 LCFA dampen intestinal Frontiers in Microbiology 11 frontiersin.org Llauradó-Calero et al. 10.3389/fmicb.2022.982712 inflammation. In terms of differential microbial populations, n- Posthaus et al., 2020). Although C. perfringens infection causes 3 LCFA modified one phylum, 15 families, and 27 genera in feces diarrhea, low weaning weights, and pre-weaning mortality, from suckling piglets. no differences in body weight nor mortality were observed Among the observed changes in the fecal microbiota of between treatments for suckling piglets at weaning. Within the suckling piglets, the phylum Verrucomicrobia increased due same phylum, we also observed an increase in the abundance to the increase in the Akkermansia genus with its tentative of Streptococcus, which contains some species described species A. muciniphila. A previous study with mice also reported as probiotics and has been related to the improvement of an increase in A. muciniphila after fish oil supplementation colostrum quality, milk quality and quantity, litter size, and (Caesar et al., 2015). Moreover, another study in healthy humans piglet vitality, and body weight (Knecht et al., 2020). In described an increase in the family Akkermansiaceae in an n- addition, Lactobacillus species (OTUs tentatively identified as 3 LCFA-treated group (Watson et al., 2018). A. muciniphila is L. delbrueckii and Lactobacillus mucosae) were also increased the most common species of Verrucomicrobia and colonizes the in the feces of n-3 LCFA suckling piglets. Previous reports have mucus layer acting as a mucin degrader (Ottman et al., 2017). described an immunomodulatory effect of L. mucosae (Ryan It is well established that it plays a crucial role in supplying et al., 2019), and that the oral administration of L. delbrueckii mucin-derived nutrients to other members of the gut microbiota improves intestinal integrity, stimulates the intestinal immune that are unable to degrade the mucin layer by themselves response, and alleviates intestinal oxidative damage in piglets (Tailford et al., 2015). This mucin-degrading capacity makes (Li Y. et al., 2019; Chen et al., 2020). Moreover, Yang et al. A. muciniphila a modulator for gut homeostasis improving (2017) described that many beneficial bacteria belonging to and regulating the gut barrier function (Guo et al., 2017). the Firmicutes phylum, such as Enterococcus, Streptococcus, In addition, its presence in the feces of highly feed-efficient Lactobacillus, and Clostridium, were reduced in diarrheic animals (Gardiner et al., 2020) and its possible role as the piglets. In the current study, these beneficial genera and host immune system modulator (Crespo-Piazuelo et al., 2019) pertaining species were increased by dietary n-3 LCFA. have also been previously described. Other studies propose Relevant modifications of bacterial populations belonging that excessive mucin degradation may facilitate the access of to phyla Bacteroidetes and Proteobacteria by dietary n-3 LCFA pathogens to the mucosa (Ganesh et al., 2013). However, in the should also be noted in the feces suckling piglets. Regarding current study, no differences in LPS concentration in the plasma Bacteroidetes, n-3 LCFA increased the Bacteroides genus, which of suckling piglets at weaning, as a gut barrier integrity marker, is one of the core bacterial genera of pigs’ microbiota and were observed between n-3 LCFA and control diets. is reported to be found in more than 90% of healthy pigs Within the important bacterial populations of the of different ages (Luo et al., 2022). Moreover, as described Firmicutes phylum that were modified by n-3 LCFA in the above for A. muciniphila, genus Bacteroides is also considered feces of suckling piglets, we observed a decrease in the family to be a mucin glycan degrader (Bell and Juge, 2021) and its Ruminococcaceae and the increase of family Clostridiaceae, low abundance has been associated with post-weaning diarrhea which are two of the core families in the swine gastrointestinal (Ren et al., 2022). It should be mentioned that n-3 LCFA also tract (Holman et al., 2017). Decreased Ruminococcaceae was due to the decrease of genera Ruminococcus (tentatively identified decreased Prevotella copri species, which are present during as R. gnavus) and Gemmiger, and increased Clostridiaceae due lactation at low abundances but increase drastically upon to the increase of the genus Clostridium and, specifically, the weaning (Amat et al., 2020). A high abundance of P. copri increases of OTUs identified as C. innocuum, C. cadaveris, has recently been related to hosting chronic inflammation and C. perfringens. On the one hand, as already described, responses resulting in excessive fat accumulation in pigs (Chen Ruminococcaceae is associated with anti-inflammatory effects et al., 2021). In terms of Proteobacteria, n-3 LCFA increased (Liu et al., 2019). Conversely, R. gnavus has been associated the Pectobacteriaceae and Enterobacteriaceae families mainly with a pro-inflammatory role (Hall et al., 2017; Henke et al., due to the increases of genus Brenneria and Escherichia, 2019). On the other hand, members of the family Clostridiaceae respectively, modifications that match those observed in milk. are also associated with the production of butyrate, so they Enterobacteriaceae consists of a set of genera that colonize the can also contribute to decreasing inflammation in the gut of intestinal microbiota and includes commensal microbiota as the host (Holman et al., 2017). However, species C. innocuum, well as pathogens (Schierack et al., 2007). This family contains C. cadaveris, and C. perfringens can become pathogenic causing LPS-producing bacteria and Escherichia coli species, which can infections (Crum-Cianflone, 2009; Gupta et al., 2020; Posthaus cause diarrhea and infections in both humans and animals et al., 2020). Concretely, C. perfringens can cause severe, acute, (Schierack et al., 2007; Costantini et al., 2017). However, in the and necrotic enteritis in humans and livestock, particularly present study, despite Escherichia being increased in the feces of in neonatal pigs (Posthaus et al., 2020). The pathogenicity n-3 LCFA suckling piglets, no increase in E. coli was detected of C. perfringens is given by toxin-α and toxin-β, which are and as already mentioned, there were no differences in piglets’ generated by C. perfringens type A and C (Baker et al., 2010; LPS plasma levels. Frontiers in Microbiology 12 frontiersin.org Llauradó-Calero et al. 10.3389/fmicb.2022.982712 According to Lauridsen (2020), colostrum, milk and oral- genera Akkermansia and Bacteroides, and different species of fecal transmission play a crucial role in the acquisition and Lactobacillus, which may contribute to the achievement of a establishment of the microbiota of newborn piglets. Some gut anti-inflammatory microbiota. In addition, it can also be correlations were observed between lactating sows’ feces and concluded that some of these modifications were positively milk, milk and suckling piglets’ feces, and lactating sows’ feces correlated among the feces of lactating sows, their milk, and the and suckling piglets’ feces showing that the changes observed feces of the suckling piglets. Milk stands out as the factor with in the former due to the inclusion of dietary n-3 LCFA may more bacterial populations correlated with both, lactating sows’ be transferred and have an impact on the latter. First, between and suckling piglets’ feces. lactating sows’ feces and milk, the family Ruminococcaceae and genus Ruminococcus in lactating sows were positively correlated with the same family and genus in milk, but also with the family Spirochaetaceae, and genera Anaerostipes Data availability statement and Treponema. Second, the genera Akkermansia, Bacteroides, The datasets presented in this study can be found in Flintibacter, and Parabacteroides in milk correlate positively online repositories. The names of the repository/repositories with the genus Parabacteroides in suckling piglets’ feces. and accession number(s) can be found below: https://www.ebi. Concretely, different OTUs belonging to these four previous ac.uk/ena, PRJEB53326. genera in milk were positively correlated with Parabacteroides distasonis in suckling piglets’ feces, which is described as immunomodulatory health-promoting bacteria (Kverka et al., 2011). Moreover, positive correlations between lactating sows’ Ethics statement feces and suckling piglets’ feces were also observed. OTUs such as Olsenella scatoligenes, Erysipelatoclostridium ramosum, This animal study was reviewed and approved by IRTA’s Peptostreptococcus stomatis, A. muciniphila, and L. delbrueckii Ethical Committee on Animal Experimentation and Generalitat on feces from lactating sows were positively correlated with the de Catalunya (project no: 10294). Bacteroides species Bacteroides stercoris and Bacteroides fluxus in feces from suckling piglets. Correlations between species are reported in Supplementary Figure 1. Therefore, milk appears to Author contributions be the type of sample with more bacterial populations positively correlated with those in the feces of lactating sows or suckling IB, RL, DT, EE-G, and NT contributed to conception and piglets, suggesting its key role in microbial transfer from sow design of the study. EL-C, ErC, EmC, MB, and NT performed to piglets. Finally, it is important to mention that the study of the methodology and statistical analysis. EL-C wrote the first correlations was performed using samples that were collected at draft of the manuscript. ErC and EmC wrote sections of the the same time and although the transference of microbiota is manuscript. All authors contributed to manuscript revision, not an immediate process, sampling was carried out when both, read, and approved the submitted version. sows and piglets, had been eating for several days the same diet and it could be assumed that results are representative of the correlations among the different types of samples. Nevertheless, a future study evaluating microbial transference considering a Funding time window between samplings may be of interest. This research was supported by the National Institute for Agricultural and Food Research and Technology (INIA) (Project RTA2017-00086-C02-01) and Eudald Llauradó-Calero Conclusion obtained an INIA grant (PRE2018-086726) to carry out this research. The inclusion of fish oil as a source of n-3 LCFA in sow diets influences the microbiota of the feces of gestating sows, their milk, and the feces of suckling piglets, while no effects were observed in colostrum or the feces of lactating sows. Acknowledgments The largest impact of n-3 LCFA supplementation is observed in the feces of suckling piglets, which are young animals that We acknowledge all the farm staff from the IRTA Mas Bover are in the process of acquiring microbiota. Concretely, n-3 facilities to make this trial possible. We also thank NP from IRTA LCFA increased piglets’ fecal microbial diversity and the relative Mas Bover laboratory for her contribution to the LPS analysis abundance of beneficial bacteria such as the mucin-degraders and JFMB from ADM for their laboratory work. Frontiers in Microbiology 13 frontiersin.org Llauradó-Calero et al. 10.3389/fmicb.2022.982712 Conflict of interest organizations, or those of the publisher, the editors and the reviewers. 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