A non-invasive tool to collect small intestine content in post weaning pigs: validation study

The Capsule for Sampling (CapSa) is an ingestible capsule that collects small intestine content while transiting through the natural digestive pathway. In this study, 14 Swiss Large White pigs weighing less than 12 kg (Category < 12 kg) and 12 weighing between 12 and 20 kg (Category [12–20 kg]) were given two CapSas and monitored for three days. The animals were euthanized for post-mortem sampling, allowing us to directly obtain gut microbiota samples from the gastrointestinal tract. This post-mortem approach enabled a direct comparison between the microbial content from the gut and the samples collected via the CapSas, and it also facilitated precise identification of the CapSas’ sampling sites within the gastrointestinal tract. For the category under 12 kg, only 2.3% of the administered CapSas were recovered from the feces. In contrast, in the 12–20 kg category, 62.5% of the CapSas were successfully retrieved from the feces within 48 h. Of these recovered CapSas, 73.3%—equating to 11 capsules from eight pigs—had a pH > 5.5 and were therefore selected for microbiome analysis. Bacterial composition of the CapSas was compared with that of the three segments of the small intestine, the large intestine and feces of the corresponding pig. The results were tested using a PERMANOVA model (Adonis) including sample type as a factor, and then pairwise comparisons were made. The bacterial composition found in the CapSas differed from that of the large intestine and feces (P < 0.01), while it did not differ from the first segment of the small intestine (P > 0.10). This study provides evidence that the CapSa effectively samples the intestinal microbiota from the upper section of the small intestine in post-weaning pigs. Furthermore, it was found that the collection of CapSas could only be successfully achieved in pigs classified within the heavier weight category.

The link between gut microbiota and pig health is well-documented 1,2 , with gut microorganisms playing crucial roles in immunity and nutrient digestion 3,4 .While existing studies predominantly examine fecal microbiota 5 , these do not accurately reflect the microbiota in other digestive tract segments like the small intestine 6,7 , where the composition varies by location and pig age 8,9 .This variation is significant, especially in the small intestine, for nutrient digestion and immune system development 10 .Understanding host-microbiota interactions requires studying the gut microbiota's spatio-temporal changes, emphasizing the need for precise, repeatable sampling methods.
There are several methods for sampling gut microbiota.Post-mortem sampling gives access to all segments of the digestive tract, but can only be performed once on the same individual.Fecal sampling, on the other hand, is non-invasive and can be repeated several times on the same individual, but only represents the fecal microbiota 11 .Additionally, there are other sampling methods that are considered highly invasive like endoscopy, biopsies and cannulated animals.Endoscopy allows multiple sampling of intestinal contents and tissues (biopsies), but can only be performed under general anaesthesia.Finally, some studies have used cannulated animals for repeated sampling of the small intestine content 12,13 .For ethical and practical reasons, these last two methods are not always easily feasible.In 2020, Tang et al. highlighted the need for new sampling methods that would allow multiple, non-invasive samplings of intestinal contents 14 .
Recent advancements in ingestible medical devices designed to traverse the digestive tract have enabled the detailed sampling and examination of the human gut microbiota [15][16][17] .These devices are either an osmotic pill [15][16][17] or an enteric-coated bladder 16,17 .Their sampling mechanism is activated when they reach the small intestine.Existing devices have been developed for humans and none have been successfully tested in pigs.

Characteristics of CapSas recovered from feces
Of the 16 CapSas recovered from the feces, two were broken and 12 (81.2%)had a pH > 5.5.The pH of the CapSa content was not affected (P > 0.05) by the body weight (BW) category (Fig. 2), but by the sex of the pigs (P = 0.048) and the transit time (P = 0.038).The pH of the CapSa content was < 6, when the transit time exceeded 48 h.The volume of digesta samples collected was not affected by the weight category, sex or transit time (P > 0.05).All CapSas recovered from feces within 48 h after administration and having a pH > 5.5 were considered for microbiota analysis.

Microbiota analysis
A total of eleven CapSas collected from eight pigs weighing between 12 and 20 kg were sent for microbiota analysis.From these 11 samples, bacterial DNA was successfully extracted and amplified.Thereafter, DNA sequencing was performed for all but one CapSa that did not contain sufficient DNA.
Analysis of β-diversity by the Principal Coordinate Analysis (PCoA) demonstrated a clustering of microbiota according to sample type (Fig. 3).The large intestine and feces microbiota clustered together in a first cluster, while the microbiota from samples of the CapSas and the three segments (Segment 1, 2 and 3) of the small intestine tended to form a second cluster.Based on the Adonis test, sample location and origin influenced the bacterial composition (P = 0.001).Pairwise comparisons detected differences in the bacterial composition between the CapSa contents, the contents of the three intestinal segments, the contents of the large intestine and feces (Table 2).According to this analysis, the bacterial composition of the three segments of the small intestine did not differ.There was also no difference between the bacterial composition of the large intestine and that of the feces.The microbial composition of the CapSa contents was different from that of the feces and large intestine, but not from that of Segment 1 (P = 0.32) and there was a tendency towards similarity with Segment 2 (P = 0.06) of the small intestine.It did, however, differ from that of Segment 3 (P = 0.01).A list of the ten main abundance Phylum, Family and Genera (expressed as % of total with the standard deviation) can be found as Supplementary Table S1.
Figure 4 shows the alpha diversity values for Chao1, Shannon and InvSimpson indices for each sample.We compared the alpha diversity of the CapSa samples to that of other sample types.The overall bacterial richness (Chao1) was significantly higher in CapSa samples compared to Segment 3 (P < 0.10), tended to be lower compared to fecal samples (P = 0.06) and no differences were observed for all other comparisons.Similarly, Shannon diversity was higher in CapSa samples compared to Segment 3 (P < 0.01) and tended to be lower if compared with feces (P = 0.06), while no differences were observed for all other comparisons.The InvSimpson diversity was higher in CapSa compared to Segment 3 (P = 0.02) and tended to be lower if compared with feces (P = 0.08), while no differences were observed for all other comparisons.

Discussion
This study validates the first non-invasive device to collect small intestinal content for microbiota analysis in postweaning pigs.CapSas administered orally and then retrieved in the feces collected digesta with a pH > 5.5.As the pH of the fasting stomach does not exceed 5.5 18 , CapSa's sampled the contents of a segment after the stomach.In addition, the in vitro studies showed that the majority of CapSas sample within one hour of being placed in an aqueous medium at pH = 7 19 .The in vivo sampling site is dependent on the increase of pH after passing through the stomach and its location depends on the speed of transit of the CapSa through the small intestine.Henze et al. 20 reported that it took the SmartPill ® , an indigestible capsule of a slightly larger size (26 mm × 13 mm) 2.3 to 4 h to pass through the small intestine of male Landrace pigs weighing 15-17 kg.Together with the in vitro finding we conclude that CapSa sampling site is located in the small intestine.
The stomach appears to be the only site where CapSa can get trapped as no CapSas were found outside the stomach when examining the entire digestive tract post-mortem.In addition, prolonged transit time was associated with lower pH of the sampled content.We hypothesize that due to a prolonged retention time in the Table 2. Comparisons of the bacterial compositions of the capsule contents, the three segments of the small intestine (Seg 1, 2 and 3), the large intestine and feces, using the Adonis test of Euclidean distances of clrtransformed data.SumsOfSqs, Sum of square reflecting total variance; F. Model, F test value; r 2 , r-square value, reflects grouping differences, the higher the value, the higher the grouping differences; P, P value; P adj, P values adjusted for multiple comparison using the Bonferroni correction.www.nature.com/scientificreports/stomach, CapSa fail to resist acidic conditions which activates the sampling mechanism.Therefore, we conclude that content of CapSa with a pH < 5.5 could contain gastric content.Our findings align with the conclusions of the study by Rezaei Nejad et al. 15 , which found that capsules retrieved from the stomachs of pigs had a bacterial composition similar to that of the stomach content.
Expanding on this, we considered additional factors influencing the pH of the retrieved CapSa contents like the metabolization of the microbiome inside the capsule and the aggregation of SCFAs.Short-chain fatty acids, www.nature.com/scientificreports/recognized as weak acids, are acknowledged contributors to pH modulation within the gastrointestinal tract.Notably, Hadinia et al. 21demonstrated a pH-dependent production of SCFA, with the highest and lowest amounts observed at pH 6 and pH 5, respectively.Another study by Henze et al. 22 further underscored the influence of pH on SCFA production, particularly noting that a mildly acidic pH (5.5) stimulates specific SCFA production.Although this study did not directly assess fermentation within the CapSas, the existing literature suggests that the involvement of SCFAs in acidifying the CapSa contents cannot be ruled out.Therefore, to ensure that CapSa contained digesta from the small intestine, only CapSas with a pH > 5.5 and a transit time of < 48 h were sent for DNA extraction and subsequent microbiota analysis.In our study, 11 CapSas from eight pigs met these requirements.
We then proceeded to validate the CapSa based microbiota composition.Applying the PCoA and the Adonis test, we demonstrated that the microbiota composition of the CapSa content is similar to that of Segment 1, tends to diverge from Segment 2, and is markedly distinct from that in Segment 3, large intestine and fecal samples.Previous studies have shown that the composition of microbiota varies significantly between different gastrointestinal segments and feces.Zhao et al. 6 concluded that microbial profiles in feces were different from those in the small intestine.In addition, the microbial composition in the large intestine was more similar to feces than the small intestine, even across different pigs' age 6 .Similarly, Adhikari et al. 23 demonstrated the difference in microbiota composition between digesta samples from jejunum and colon on weaned piglets 6,23 .In the present study, the comparisons of the bacterial compositions of the three segments of the small intestine and feces showed a completely different composition.This further confirms that feces is not representative of the small intestinal microbiota.
Overall, it can be observed how bacterial richness and diversity tend to increase going from the small intestine to feces, these results are in line to what was observed in other studies 7,24 .In all three indices, the alpha diversity of the CapSa content did not differ from that observed in Segment 1 and 2 but was significantly higher than that in Segment 3. The similarity in alpha diversity between the CapSa content and Segment 1 indicates that the CapSa sample accurately mirrors the species diversity observed in Segment 1.
As the bacterial density in the lumen is higher than at or within the mucosa 25 , most mucosa-associated bacteria are represented in the luminal contents 26 and many metabolites of interest are in the lumen.Due to its opening and closing mechanism and its movement via natural peristaltic motions, we hypothesized that CapSa samples luminal bacteria.These capsules therefore provide a non-invasive alternative to sample the content of the small intestine in pigs, which can be used for any laboratory analysis that can be performed with ~ 150µL.
To date, only a few sampling capsules were able to collect intestinal content.The sampling mechanism of the pill developed by Rezaei Nejad et al. 15 was tested in weaned pigs and in macaques.They validated their sampling device in vivo by comparing the microbial composition profile of the capsule's sample to that of the matrix from which the capsule was retrieved.In their study, the bacterial composition of pills found in pigs stomach clustered with the stomach contents, while those found in feces clustered with the fecal microbial profile.The results in macaques were quite different, and the bacterial microbiome collected by the pill retrieved in feces was clearly different from feces.The authors concluded the pill was able to sample the regions of the gut upstream of the colon with quite distinct microbiome populations compared with the feces.Shalon et al. 17 developed pill prototypes that can sample four different sites in human small intestine, from the duodenum to jejunum.To validate their device as a sampling tool to collect small intestine content, they attached one pill to a capsule endoscope and visualized successful video sampling in one human.They further confirm their results by observing differences between pills and saliva/stool samples, specifically in microbiota composition, prophage induction, protein abundance and bile acids profile.
With the aim of using CapSa for pigs, a standardised protocol was necessary to administer the capsules.Indeed, pig's gastric emptying is very slow, and only small amounts of stomach content leave the stomach at once 27 .The speed of gastric emptying is highly variable between pigs, and large solid particles (> 1 cm) can remain in the stomach for several days 20 .The reason for this delayed emptying is anatomical.Indeed, pig's stomach has a very pronounced "C" shape, and the gastric cardia is very close to the pylorus 27 .In addition, a transverse pyloric fold, called the torus pyloricus, is located right before the pylorus and serves as a "gate-keeper" to prevent any large particle to enter the small intestine 28 .To overcome the anatomical limitations, the administration protocol consisted in providing a liquid feed to limit gastrointestinal load, and a prokinetic to reinforce gastric contractions.A prokinetic is a substance that amplifies and coordinates the gastrointestinal muscular contractions to facilitate the transit 29 .Despite this protocol, pig's BW still affected the percentage of CapSas found in the stomach, and consequently the percentage of CapSas recovered from feces.The smaller the piglet, the more likely the CapSa became stuck in the stomach.This explains why only pigs over 12 kg could be successfully sampled.As a consequence, CapSa is not an effective device in pigs with a bodyweight below 12kg since its retrieval in feces is impossible due to anatomical reasons.
The transit time did not distort bacterial composition.Indeed, CapSa content still had similar bacterial composition to Segment 1 of the small intestine, despite < 48h of transit under body temperature (38°C).Similarly, Shalon et al. 17 demonstrated that there were no major changes in microbiota composition if transit (incubation) did not exceed 58 h.
This study validates the first non-invasive device for the collection and analysis of intestinal microbiota in post-weaning pigs with a bodyweight above 12 kg.A standardised protocol has also been established for successful deployment of the CapSas in pigs.This new tool opens new perspectives to study the gut physiology.
Further studies will be conducted to validate CapSa in fattening pigs as well as the effect of the protocol on the small intestine microbiota.

Animals and rearing conditions
For the study, 26 Swiss Large White pigs with BWs ranging from 6.4 to 20.0 kg were used.Fourteen weighed less than 12 kg (category < 12kg) and 12 weighed between 12 and 20 kg (category [12-20 kg]) (Table 3).Pigs of Category < 12kg were housed in groups of four and pigs of category [12-20kg] were housed in groups of two.All pigs had ad libitum access to a standard starter diet formulated to meet the nutritional requirements of postweaning pigs 30 (see Supplementary Table S2).Water was available ad libitum and distributed via nipple drinkers.The pens (total surface area of 4.47 m 2 ) were specially designed to collect the CapSas by minimizing the slatted area and reducing the openings of the slatted area to a size smaller than the CapSa diameter.

Description of the capsule (CapSa)
The capsule studied is 21.7 mm long with a diameter of seven mm, corresponding to a size 0 hard capsule.CapSa opens, collects the sample and closes depending on the physico-chemical properties of its environment.It moves along the digestive tract purely passively, and the speed at which it passes depends entirely on intestinal peristalsis.CapSa can collect a maximum of 400 µL.
CapSa is designed to operate as follows: Once ingested, it moves through the stomach and reaches the small intestine, where it opens to collect a sample.After collection, the CapSa closes, continues its journey through the large intestine, and is finally expelled with the stool.This single-use device is specifically designed to collect fluid samples from the intestines for later ex vivo analysis.As illustrated in Fig. 5, the CapSa is composed of two main components: a dissolvable exterior with an enteric coating and a 3D printed bottom part.The CapSa is engineered to open when it encounters a pH level greater than 6, as the upper part dissolves, permitting intestinal fluids to enter.The entering intestinal content triggers the plunger to expand, which draws luminal content into the CapSa's inner chamber.The CapSa is designed to automatically seal once the plunger mechanism is fully extended.In vitro results show that CapSa can withstand two hours in an acidic-aqueous medium (pH < 3), and then samples within an hour of being transferred to an aqueous medium at pH = 7 19 .

Preparation of the animals and administration of CapSa
Two days prior to CapSa administration, three measures were taken to reduce intestinal load and shorten transit time.Firstly, two days before administering the CapSa (-2d), pigs were fed the starter diet in liquid form (ratio 1 kg of starter diet mixed with 2 L of water) and straw was removed from the pens.Secondly, one day before administering the CapSa (-1d) pigs had access to only half of their ration of feed and the feed was removed 12 h before capsules administration.Thirdly, to increase gastric emptying and thus facilitate CapSa transit through the stomach, 0.16 ± 0.015 mg/kg BW of prucalopride (Resolor ® , Takeda Pharma AG, Glattpark, Switzerland) was administered orally via an oesophageal probe 40 min prior to administration.Prucalopride is a 5-HT 4 serotonin agonist, which stimulates gastrointestinal peristalsis and increases gastric emptying [31][32][33][34] .
On the day of administration (0d), each pig received 2 CapSas.The capsules were administered by oesophageal sondage, while the pigs were kept in a sling adapted to their weight.A 10 mL bolus of orange juice was then

Figure 1 .
Figure 1.Time (h) of transit of CapSas found in feces.Time is calculated as the difference between the time of administration and the time of recovery.

Figure 2 . 3 Figure 3 .
Figure 2. pH and volume (µL) of CapSa contents as a function of transit time (h) and pig weight category.Weight category had no effect (P = 0.14) on the pH of the contents of capsules found in feces (a), but transit time strongly influenced pH (P = 0.038), with pH falling below six when transit time exceeded 48 h (b).The volume samples collected was not affected by weight category (P = 0.44), sex (P = 0.11) or transit time (P = 0.59) (c, d).

Figure 4 .
Figure 4. Box plots showing shows alpha diversity values for Chao1, Shannon and InvSimpson for each sample.Only p values < 0.10 are shown.

Table 1 .
Outcomes of capsule administration by weight category.SEM, Standard error of the mean; P, P value of the fixed effect.

Table 3 .
Characteristics of pigs included..BW, Body Weight; SD, Standard Deviation; %M, Percentage of castrated males; %F, Percentage of females.All experimental procedures were in compliance with Swiss animal welfare guidelines and were approved (No. 2021-39-FR) by the Cantonal Veterinary Office of Fribourg (Switzerland).All methods are reported in accordance with the ARRIVE guidelines.