**2. Results**

#### *2.1. Characterization of Seaweed Extracts*

The LAM extract was obtained from *Laminaria Digitata* and contained 653.2 g of laminarin per kg DM, 190 g/kg fucoidan per kg DM, 5 g/kg phlorotannin per kg DM, 51 g/kg mannitol per kg DM, 40 g/kg alginates per kg DM (Table 1).


**Table 1.** Biochemical composition of the extracts obtained following extraction and purification.

The FUC rich extract was obtained from *Ascophyllum nodosum* and contained 441 g of fucoidan per kg DM, 25.9 g laminarin/kg DM, 135g alginates/kg DM, 43.8 g mannitol/kg DM, 34.8 g phlorotannin's/kg DM.

## *2.2. Animal Performance*

The average body weights of the pigs on the SWE diets were the same as the control group at the end of the experimental period ( *P* > 0.05; Table 2). While no significant effects of dietary supplementation with either LAM or FUC were identified for measures of ADFI or ADG compared to the control group ( *P* > 0.05), pigs fed the FUC supplemented diet had lower G:F ratio compared to the control ( *P* < 0.05; Table 2).


**Table 2.** The effect of supplementation with either laminarin or fucoidan on animal performance.

\*ADFI: average daily feed intake; ADG: average daily gain; G:F: gain to feed ratio; a,b values with different superscripts differ significantly (*P* < 0.05); A total of seven replicates were used per treatment group (replicate = pen, 3/pigs/pen).

#### *2.3. Volatile Fatty Acids*

Pigs on the LAM diet had the highest concentrations of total volatile fatty acid (VFA) and butyrate compared to the FUC supplemented group and the basal group (*P* < 0.05; Table 3).

**Table 3.** The effect of supplementation with either laminarin or fucoidan on volatile fatty acids (VFAs) produced in colonic digesta on d35 post-weaning.


Ace:Pro: Acetate to propionate ratio, Seven replicates per treatment used; a,b,c, values with different superscripts differ significantly (*P* < 0.05); A total of seven replicates were used per treatment group (replicate = pig).

#### *2.4. Differential Abundance Analysis of Bacterial Taxa in Colonic Digesta and Alpha Diversity Analysis*

Next-generation sequencing of the 16S rRNA gene using the Illumina Miseq platform was conducted to examine the impact of feeding the experimental diets on the intestinal microbiome. This analysis assessed the impact on measures of diversity (Table 4) and differential abundance analysis that was conducted at the taxonomic levels of Phylum, Family and Genus. The analysis of all taxa is presented in the Supplementary Tables (S1–S3) while significant data are presented in Table 5.

**Table 4.** Effects of laminarin and fucoidan supplementation on measures of alpha diversity in colonic digesta.


a,b values with different superscripts differ significantly (*P* < 0.05); A total of seven replicates were used per treatment group (replicate = pig).


**Table 5.** Assessment of microbial composition in colonic digesta.

a,b,c values with different superscripts differ significantly (*P* < 0.05); \* A total of seven replicates were used per treatment group (replicate = pig).

Pigs fed the basal diet had the highest alpha diversity of the three experimental groups based on Observed, Chao1, ACE, Shannon and Fisher measures of alpha diversity (*P* < 0.05). The LAM and FUC supplemented pigs had similar levels of alpha diversity.

**Phylum:** Pigs on the LAM supplemented diets had increased abundance of Firmicutes and Spirochaetes compared to pigs fed the basal and FUC supplemented diets (*P* < 0.05). Pigs fed the FUC supplemented diet had the highest abundance of Proteobacteria compared to both the basal and LAM diets (*P* < 0.05) with pigs fed a LAM supplemented diet having lower abundance of Proteobacteria compared to the basal diet (*P* < 0.05).

**Family:** Pigs on the LAM supplemented diets had lower *Campylobacteraceae* and increased *Spirochaetaceae* compared to the other two groups (*P* < 0.05) and increased *Ruminococcaceae* compared to the FUC supplemented group (*P* < 0.05).

**Genus:** Pigs on the FUC supplemented diet had lower *Faecalbacterium* and higher *Campylobacter* compared to pigs on both the LAM and basal diets (*P* < 0.05). Pigs on the LAM supplemented diet had lower *Campylobacter* compared to the other two diets (*P* < 0.05). Pigs on the basal diet had increased *Alloprevotella* compared to pigs fed the SWE supplemented diets. LAM supplementation increased *Roseburia* compared to the pigs fed the control diet (*P* < 0.05), while also increasing *Treponema* compared to pigs fed both the basal and the pigs fed the diet supplemented with FUC (*P* < 0.05).

#### *2.5. Differential Expression Analysis of Genes Related to Nutrient Absorption and the Intestinal Immune Response*

The Nanostring nCounter technology was employed to examine the effect of LAM and FUC supplementation on the expression of genes related to intestinal health and functionality. The expression profile of 32 genes in the small intestine were measured, as described in the materials and methods. All gene expression data is presented in Supplementary Tables S4–S6 while significant data is presented in Table 6.

2.5.1. Differential Expression Analysis of Digestive Enzyme and Nutrient Transporter gene expression

Duodenum: FUC supplementation increased the expression of *SLC6A19* compared to LAM supplementation, while FUC supplementation increased the expression of *CNDP1* compared to both LAM supplementation and the basal group (*P* < 0.05). Pigs fed the basal diet had increased expression of *SLC16A10* compared to both LAM and FUC supplementation.

Jejunum: LAM supplementation increased the expression of *MCT1/SLC16A1* compared to FUC supplementation and the basal group (*P* < 0.05).


**Table 6.** Effect of laminarin or fucoidan inclusion on the gene expression of nutrient transporters and immune markers in the duodenum, jejunum and ileum.

a,b values with different superscripts differ significantly (*P* < 0.05); A total of seven replicates were used per treatment group (replicate = pig).

Ileum: FUC supplementation decreased the expression of *SLC2A2*, *GCG*, *FABP2*, *SLC5A1* and *SI* compared to the basal group (*P* < 0.05). Pigs fed the basal diet had increased expression of *CNDP1* compared to the FUC and LAM supplemented groups (*P* < 0.05).

#### 2.5.2. Differential Expression of Markers of Immune Response and Intestinal Integrity

Jejunum: Pigs on the FUC supplemented diet had increased expression of *CXCL8* compared to both LAM supplementation and the basal diet (*P* < 0.05). Pigs on the LAM supplemented diet had reduced *MUC1* compared to the basal and LAM supplemented diet (*P* < 0.05). Pigs on the basal diet had higher expression of *OCLN* compared to pigs fed the FUC and LAM supplemented diets (*P* < 0.05).

Ileum: pigs fed the FUC supplemented diet had increased expression of *TGFB1* and *TNF* compared to the control and LAM groups (*P* < 0.05). Supplementation with FUC decreased expression of *CLDN5*, *CLDN3* and *OCLN* compared to the basal diet (*P* < 0.05).
