Metabarcoding of the 16S rRNA gene is commonly used to characterize microbial communities, by estimating the relative abundance of microbes. Here, we present a method to retrieve the concentrations of the 16S rRNA gene per gram of any environmental sample using a synthetic standard in minuscule amounts (100 ppm to 1% of the 16S rRNA sequences) that is added to the sample before DNA extraction and quantified by two quantitative polymerase chain reaction (qPCR) reactions. This allows normalizing by the initial microbial density, taking into account the DNA recovery yield. We quantified the internal standard and the total load of 16S rRNA genes by qPCR. The qPCR for the latter uses the exact same primers as those used for Illumina sequencing of the V3‐V4 hypervariable regions of the 16S rRNA gene to increase accuracy. We are able to calculate the absolute concentration of the species per gram of sample, taking into account the DNA recovery yield. This is crucial for an accurate estimate as the yield varied between 40% and 84%. This method avoids sacrificing a high proportion of the sequencing effort to quantify the internal standard. If sacrificing a part of the sequencing effort to the internal standard is acceptable, we however recommend that the internal standard accounts for 30% of the environmental 16S rRNA genes to avoid the PCR bias associated with rare phylotypes. The method proposed here was tested on a feces sample but can be applied more broadly on any environmental sample. This method offers a real improvement of metabarcoding of microbial communities since it makes the method quantitative with limited efforts.

Dietary fiber content and composition affect microbial composition and activity in the gut, which in turn influence energetic contribution of fermentation products to the metabolic energy supply in pigs. This may affect feed efficiency (FE) in pigs. The present study investigated the relationship between the fecal microbial composition and FE in individual growing-finishing pigs. In addition, the effects of diet composition and sex on the fecal microbiome were studied. Fecal samples were collected of 154 grower-finisher pigs (3-way crossbreeds) the day before slaughter. Pigs were either fed a diet based on corn/soybean meal (CS) or a diet based on wheat/barley/by-products (WB). Fecal microbiome was characterized by 16S ribosomal DNA sequencing, clustered by operational taxonomic unit (OTU), and results were subjected to a discriminant approach combined with principal component analysis to discriminate diets, sexes, and FE extreme groups (10 high and 10 low FE pigs for each diet by sex-combination). Pigs on different diets and males vs. females had a very distinct fecal microbiome, needing only 2 OTU for diet (P = 0.020) and 18 OTU for sex (P = 0.040) to separate the groups. The 2 most important OTU for diet, and the most important OTU for sex, were taxonomically classified as the same bacterium. In pigs fed the CS diet, there was no significant association between FE and fecal microbiota composition based on OTU (P > 0.05), but in pigs fed the WB diet differences in FE were associated with 17 OTU in males (P = 0.018) and to 7 OTU in females (P = 0.010), with 3 OTU in common for both sexes. In conclusion, our results showed a diet and sex-dependent relationship between FE and the fecal microbial composition at slaughter weight in grower-finisher pigs.

Microbiota play an important role in total tract nutrient digestion, especially when fibrous diets are fed to pigs. This study aimed to use metagenomics to predict faecal nutrient digestibility in grower‐finisher pigs. The study design consisted of 160 three‐way crossbreed grower‐finisher pigs (80 female and 80 male) which were either fed a diet based on corn/soybean meal or a more fibrous diet based on wheat/barley/by‐products. On the day before slaughter, faecal samples were collected and used to determine faecal digestibility of dry matter, ash, organic matter, crude protein, crude fat, crude fibre and non‐starch polysaccharides. The faecal samples were also sequenced for the 16S hypervariable region of bacteria (V3/V4) to profile the faecal microbiome. With these data, we calculated the between‐animal variation in faecal nutrient digestibility associated with variation in the faecal microbiome, that is the “microbiability”. The microbiability values were significantly greater than zero for dry matter, organic matter, crude protein, crude fibre and non‐starch polysaccharides, ranging from 0.58 to 0.93, as well as for crude fat with a value of 0.37, but not significantly different from zero for ash. Using leave‐one‐out cross‐validation, we estimated the accuracy of predicting digestibility values of individual pigs based on their faecal microbiota composition. The accuracies of prediction for crude fat and ash digestibility were virtually 0, and for the other nutrients, the accuracies ranged from 0.42 to 0.63. In conclusion, the faecal microbiota composition gave high microbiability values for faecal digestibility of dry matter, organic matter, crude protein, crude fibre and non‐starch polysaccharides. The accuracies of prediction are relatively low if the interest is in precisely predicting faecal nutrient digestibility of individual pigs, but are promising from the perspective of ranking animals in a genetic selection context.

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1 -General introduction 2 -Variation in faecal digestibility values related to feed efficiency traits of grower-finisher pigs 3 -Fecal microbial composition associated with variation in feed efficiency in pigs depends on diet and sex 4 -Prediction of nutrient digestibility in grower-finisher pigs based on faecal microbiota composition 5 -Between animal variation in dietary nitrogen and amino acid efficiency in grower-finisher pigs 6 -Combining faecal microbiome, serum metabolome, and host genome for phenotypic prediction of feed efficiency in grower-finisher pigs 7 -General discussion Summary Résumé Curriculum Vitae Acknowledgements General introduction 2 Variation in faecal digestibility values related to feed efficiency traits of grower-finisher pigs

Exploring factors that might affect nitrogen (N) efficiency in pigs could support the development of precision feeding concepts. Therefore, an experiment was conducted to determine the effects of birth weight (BiW) on N retention, N efficiency, and concentrations of metabolites in plasma and urine related to N efficiency in male pigs of 14 wk of age. BiW of the low BiW (LBW) and high BiW (HBW) pigs was 1.11 ± 0.14 and 1.79 ± 0.12 kg, respectively. Twenty LBW and 20 HBW pigs were individually housed in metabolism cages and were subjected to an N balance study in two sequential periods of 5 d, after an 11-d adaptation period. Pigs were assigned to a protein adequate (A) or protein restricted (R, 70% of A) regime in a change-over design and fed restrictedly 2.8 times the energy requirements for maintenance. Nontargeted metabolomics analyses were performed in urine and blood plasma samples. The N retention in g/d was higher in the HBW than in the LBW pigs (P < 0.001). The N retention in g/(kg BW0.75·d) and N efficiency (= 100% × N retention / N intake), however, were not affected by BiW of the pigs. Moreover, fecal digestibility of N and urinary concentration of N and urea were not affected by BiW of the pigs. The concentration of insulin (P = 0.08) and insulin-like growth factor-1 (IGF-1;P = 0.05) in blood plasma was higher in HBW pigs, whereas the concentration of α-amino N tended to be lower in HBW pigs (P = 0.06). The LBW and HBW pigs could not be discriminated based on the plasma and urinary metabolites retrieved by nontargeted metabolomics. Restricting dietary protein supply decreased N retention (P < 0.001), N efficiency (P = 0.07), fecal N digestibility (P < 0.001), urinary concentration of N and urea (P < 0.001), and concentration of urea (P < 0.001), IGF-1 (P < 0.001), and α-amino N (P < 0.001) in blood plasma. The plasma and urinary metabolites differing between dietary protein regime were mostly amino acids (AA) or their derivatives, metabolites of the tricarboxylic acid cycle, and glucuronidated compounds, almost all being higher in the pigs fed the A regime. This study shows that BiW affects absolute N retention but does not affect N efficiency in growing pigs. Therefore, in precision feeding concepts, BiW of pigs should be considered as a factor determining protein deposition capacity but less as a trait determining N efficiency.

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The effects of birth weight (BiW) (low BiW (LBW) vs high BiW (HBW)) and estimated breeding value for protein deposition (EBV) (low EBV (LBV) vs high EBV (HBV)) on N retention, N efficiency and concentrations of metabolites in plasma and urine related to N efficiency in growing pigs were studied. At an age of 14 weeks, 10 LBW-LBV (BiW: 1.07 + 0.09 (SD) kg; EBV: -2.52 + 3.97 g/d, compared to an average crossbred pig with a protein deposition of 165 g/d), 10 LBW-HBV (BiW: 1.02 + 0.13 kg; EBV: 10.47 + 4.26 g/d), 10 HBW-LBV (BiW: 1.80 + 0.13 kg; EBV: -2.15 + 2.28 g/d), and 10 HBW-HBV (BiW: 1.80 + 0.15 kg; EBV: 11.18 + 3.68 g/d), male growing pigs were allotted to the experiment. The pigs were individually housed in metabolism cages and were subjected to a N balance study in two sequential periods of 5 d, after a 11-d dietary adaptation period. Pigs were assigned to a protein adequate (A) or protein restricted (R, 70% of A) regime in a change-over design. Pigs were fed 2.8 times the energy requirements for maintenance. Non-targeted metabolomics analyses were performed in urine and blood plasma samples. The N retention (in g/d) was higher in the HBW than in the LBW pigs (P < 0.001). The N retention (in g/(kg BW 0.75.d)) and N efficiency, however, were not affected by BiW of the pigs. The N retention (P = 0.04) and N efficiency (P = 0.04) were higher in HBV than in LVB pigs on the A regime, but were not affected by EBV in pigs on the R regime. Restricting the dietary protein supply with 30% decreased the N retention (P < 0.001) but increased the N efficiency (P = 0.003). Non-targeted metabolomics showed that a hexose, free amino acids (AA) and lysophosphatidylcholines were the most important metabolites in plasma for the discrimination between HBV and LBV pigs, whereas metabolites of microbial origin contributed to the discrimination between HBV and LBV pigs in urine. This study shows that BiW does not affect N efficiency in later life of pigs. Nitrogen efficiency and N retention were higher in HBV than in LBV pigs on the A regime, but similar in HBV and LBV pigs on the R regime. In precision feeding concepts aiming to further optimize protein and AA efficiency in pigs, the variation in EBV for protein deposition of pigs should be considered as a factor determining N retention, growth performance and N-efficiency.