Simple Summary Seaweeds contain various bioactive compounds that may influence gut microbial activity and host metabolism, but their effects in companion animals are still not well understood. In this study, healthy adult dogs were fed diets containing either Ulva sp. or Gloiopeltis tenax for 16 weeks under controlled feeding conditions. Dogs receiving the G. tenax-containing diet showed differences in several gut microbial groups, glycan-degrading enzyme activities, and serum metabolites, while nutrient digestibility and general health-related parameters remained largely unchanged. These findings provide preliminary insight into microbiome- and metabolome-associated responses to dietary seaweed supplementation in dogs and support further research on functional dietary ingredients for companion animals. Abstract Gloiopeltis tenax is a red seaweed containing diverse polysaccharides and bioactive compounds with potential functional applications in animal nutrition. However, information regarding its physiological and microbiome-associated effects in companion animals remains limited. The present study was designed as an exploratory nutritional intervention to evaluate physiological responses associated with dietary G. tenax supplementation in healthy adult dogs using an integrated framework including nutrient digestibility, glycan-degrading enzyme activity, fecal microbiome profiling, and serum metabolomics. Ten healthy adult dogs were assigned to two dietary groups receiving nutritionally balanced diets containing either Ulva sp. (CON) or G. tenax (GT) at 1% inclusion for 16 weeks under standardized feeding and housing conditions. Nutrient digestibility, fecal glycan-degrading enzyme activities, fecal microbiome composition, predicted microbial functional profiles, and serum metabolomic responses were evaluated. No significant differences were observed in nutrient digestibility, fecal score, or general health-related parameters between groups, suggesting acceptable tolerability of dietary G. tenax under the present experimental conditions. Relative abundances of several bacterial taxa differed between groups, and glycan-degrading enzyme activities showed directional changes associated with dietary treatment. PICRUSt2-based analyses suggested potential differences in predicted carbohydrate- and glycan-associated microbial functional tendencies between groups. Serum metabolomic analysis additionally revealed alterations in several amino acid- and carbohydrate-related metabolites associated with dietary intervention. Collectively, these findings provide preliminary insight into microbiome- and metabolome-associated responses to dietary G. tenax supplementation in dogs. Although limited by the exploratory nature and relatively small sample size of the present study, the integrated multi-omics approach applied here may contribute to the development of functional evaluation frameworks for companion animal dietary ingredients. Further studies with larger cohorts and expanded functional analyses are warranted. 1. Introduction Gloiopeltis tenax, a red seaweed traditionally used as a food and medicinal material, contains viscous polysaccharides and other bioactive constituents [ 9, 10]. Indeed, there is an increasing research focus on identifying novel and safe functional ingredients from diverse natural resources, such as the validation of Korean native black goat meat as a protein source [ 11] and the anti-obesity potential of barley sprouts ( Hordeum vulgare L.) as a safe dietary supplement for dogs [ 12]. With increasing interest in the microbiota-modulating potential of seaweed-derived polysaccharides, G. tenax has emerged as a candidate ingredient that may induce a positive effect on the gut microbiota. Nevertheless, integrated evaluations in dogs that jointly assess gut microbiota composition, predicted microbial functions, and host metabolic readouts following G. tenax supplementation remain limited. Importantly, microbiota changes are not limited to taxonomic shifts but may also be linked to altered metabolic capacities that influence the host metabolome [ 13, 14, 15]. Although 16S rRNA gene sequencing is widely used to profile microbial community structure, tools such as PICRUSt2 enable the inference of predicted functional potential at the gene family and pathway levels based on taxonomic profiles [ 16]. In addition, serum metabolomics using 1H nuclear magnetic resonance ( 1H NMR) can capture systemic metabolic responses to dietary interventions [ 17, 18]. Integrating microbiota composition, predicted functions, and serum metabolomic profiles may, therefore, provide a more comprehensive view of the gut–host metabolic axis in dogs [ 13, 14, 15]. 2. Materials and Methods 2.1. Animals and Experimental Design The experimental protocol was reviewed and approved by the Animal Care and Use Committee of the National Institute of Animal Science (NIAS) in Wanju, Republic of Korea (NIAS2023-0615). Ten clinically healthy adult small-breed dogs owned by NIAS were used in this experiment, including five Maltese and five Poodles (five spayed females and five neutered males; approximately three years old). Only dogs without a history of gastrointestinal disease, recent antibiotic exposure, or metabolic disorders were included in the study. Body condition score (BCS) was evaluated using a 9-point scale [ 24]. The two treatments were as follows: (1) a basal diet containing 1% green seaweed ( Ulva sp.; CON) and (2) a basal diet containing 1% red seaweed ( Gloiopeltis tenax; GT). Dogs were allocated to treatment groups (n = 5 dogs per treatment) to achieve a balanced distribution of breed, sex, body weight (BW), and BCS prior to dietary intervention. The CON group consisted of three Maltese (female:male = 1:2) and two Poodles (female:male = 1:1), whereas the GT group consisted of two Maltese (female:male = 1:1) and three Poodles (female:male = 2:1). The feeding trial was conducted for a total of four weeks. All experimental animals were individually housed indoors in pens measuring 1.7 m × 2.1 m per dog under controlled environmental conditions (22–24 °C; 12 h light/12 h dark cycle). During the experimental period, all dogs received approximately 6 h of daily outdoor activity in individual outdoor spaces (2.8 m × 2.5 m per dog) connected to the indoor facility. Animal care, feeding management, and daily monitoring were performed by the same trained personnel throughout the study period to minimize environmental variation among animals. Each dog received feed according to its individual metabolizable energy (ME) requirement (132 kcal × BW 0.75 kg/day) in accordance with the Association of American Feed Control Officials (AAFCO) recommendations [ 25]. Water was provided ad libitum. All dogs were routinely monitored under veterinary supervision throughout the experimental period, and no animals showed clinical signs requiring medical intervention during the study. 2.2. Preparation of Experimental Diets The dried G. tenax used in this study was provided by the National Institute of Fisheries Science (NIFS, Busan, Republic of Korea). All experimental diets were formulated to meet AAFCO nutrient requirements [ 25] and designed to be nutritionally equivalent ( Table 1). The 1% inclusion level was selected based on previous canine and animal nutrition studies using seaweed-derived ingredients [ 26]. Except for lard, all ingredients were obtained in powdered form from commercial sources, and no flavoring agents or preservatives were used. Experimental diets were prepared by mixing the ingredients based on the formulation, followed by steaming, molding, cutting, and drying to produce the final pellets. All experimental diets were stored in a −20 °C freezer until used and allowed to reach room temperature for 3 h before feeding. 2.3. Measurement of Feed Intake and Body Weight Throughout the experimental period, feed residue and fecal scores were recorded daily, and body weight was measured weekly. Average daily feed intake (ADFI) and body weight gain (BWG) were calculated for each period based on the recorded feed residue and BW. BCS was assessed weekly by the same evaluator using the 9-point BCS scale described by Laflamme [ 24]. Fecal scores were recorded daily by the same evaluator using a 5-point fecal scoring scale (1 = dry feces to 5 = liquid feces) and expressed as the mean weekly value during the experimental period. 2.4. Assessment of Apparent Nutrient Digestibility Nutrient digestibility was analyzed using the total fecal collection method. All feces were collected for four consecutive days beginning four days before the end of the experiment. Fecal samples were collected individually from each dog and stored at −20 °C immediately after collection until analyzed. Diet samples were also stored at −20 °C until they were used. The chemical compositions of the diets and feces were analyzed for moisture (AOAC method 934.01), crude protein (CP; AOAC method 984.13), ether extract (EE; AOAC method 920.39), crude fiber (CF; AOAC method 978.10), and crude ash (CA; AOAC method 942.05) [ 27]. The gross energy (GE) of the experimental diet and feces was determined using an adiabatic oxygen bomb calorimeter (6400 automatic isoperibol calorimeter; Parr Instrument Company, Moline, IL, USA). Nitrogen-free extract (NFE) was calculated as follows: NFE (%) = 100 − (Moisture + CP + CF + EE + CA). Amino acids were analyzed using high-performance liquid chromatography (HPLC; Shimadzu LC-10AT, Kyoto, Japan). Apparent total tract digestibility (ATTD) was calculated using the following equation: ATTD (%) = [(Amount of nutrient intake − Amount of fecal nutrient excretion)/Amount of nutrient intake] × 100. 2.5. Measurement of Fecal Glycan-Degrading Enzyme Activity Fecal glycan-degrading enzyme activity was assessed according to the protocol described by Steimle et al. [ 28]. Approximately 30~50 mg of fecal sample was homogenized in phosphate-buffered saline (PBS, pH 7.0) and subjected to sonication for cell lysis. The lysates were centrifuged, and the supernatants were collected for enzyme activity measurements. Activities of five glycan-degrading enzymes, including sulfatase (4N-S), fucosidase (4N-FP), N-acetyl- β-glucosaminidase (4N-NAG), galactosidase (4N-GalP), and glucosidase (4N-GluP), were measured using 4-nitrophenyl-linked substrates. The released 4-nitrophenol was quantified by measuring the absorbance at 405 nm using a microplate spectrophotometer (Multiskan SkyHigh, Thermo Scientific, Waltham, MA, USA), with 4-nitrophenol as the calibration standard. The total protein content of the fecal lysate was determined using the bicinchoninic acid (BCA) protein assay, and enzyme activities were normalized to this value. The results are expressed as mM of 4-nitrophenol released per mg of total protein (mM/mg protein). Statistical analyses were performed using JMP Pro 16.0 (SAS Institute Inc., Cary, NC, USA). Differences between the two groups were determined using an independent samples t-test, with statistical significance set at p 1 was used as a descriptive threshold to highlight influential variables in the model, and VIP scores were interpreted as contribution metrics for multivariate discrimination rather than as evidence of univariate significance [ 13]. Model summaries (R 2X, R 2Y, and Q 2) are reported as provided by the software. 2.11. Microbiome–Metabolite Integration For integrative analyses, fecal microbiota data were restricted to week 4 samples and summarized at the family level. To reduce sparsity, the top ten most abundant families ranked by mean relative abundance across dogs at week 4 were retained. Serum metabolites were expressed as Δ values (week 4 − day 0). Metabolites showing a between-group difference in unadjusted screening tests (Wilcoxon rank-sum test; GT vs. CON) at p 1), whereas glutamine was BH-FDR-significant and ranked prominently by VIP in the CON-associated profile. To explore microbiome–metabolite covariation, Spearman correlation analyses were conducted between week 4 family-level relative abundances and Δ metabolite values ( Table 9). Correlation p-values were adjusted for multiple testing using the BH-FDR procedure (i.e., correction applied to correlation p-values). Prevotellaceae abundance was positively correlated with Δ alanine and Δ methionine ( r = 0.62). Lachnospiraceae showed a positive correlation with Δ glutamine ( r = 0.55). Sutterellaceae was positively correlated with Δ creatine ( r = 0.48), and Fusobacteriaceae showed a negative correlation with Δ serine ( r = −0.44). These correlation patterns represent associative relationships and should be interpreted cautiously rather than as a confirmation of the causal microbial regulation of host metabolism. 3.7. Predicted Functional Profiles (PICRUSt2) To explore functional implications of the observed taxonomic shifts, microbial metabolic potential was inferred using PICRUSt2 and summarized at the MetaCyc pathway level. The top 10 glycan- and glycan-related pathways ranked by mean relative abundance are presented in Figure 3. Pathways including non-oxidative pentose phosphate, dTDP-L-rhamnose biosynthesis, peptidoglycan biosynthesis, glycogen biosynthesis and catabolism, and glucomannan degradation were among the most abundant in both groups. Although no individual pathway reached statistical significance after Wilcoxon rank-sum testing (all p > 0.05), the GT group showed numerically higher predicted abundance in gluconeogenesis and O-antigen biosynthesis pathways, whereas the CON group showed higher predicted activity in glycogen catabolism and glucomannan degradation. These exploratory predictions are consistent with the directional shifts in glycan-fermenting taxa observed in the compositional analysis and should be interpreted as hypothesis-generating in the context of this small sample size. 4. Discussion The present study evaluated physiological and microbiome-associated responses to dietary G. tenax supplementation in healthy adult dogs using an integrated framework including nutrient digestibility, glycan-degrading enzyme activity, fecal microbiome profiling, and serum metabolomics. Overall, dietary inclusion of G. tenax was associated with directional changes in several microbial taxa, glycan-related enzyme activities, and serum metabolites without adverse effects on nutrient digestibility or general health-related parameters under the present experimental conditions [ 1, 2]. 5. Conclusions Dietary inclusion of G. tenax was associated with shifts in fecal microbiome composition, glycan-degrading enzyme activity, and serum metabolomic profiles in healthy adult dogs under controlled feeding conditions. Several bacterial taxa, glycan-related enzyme activities, and amino acid- and carbohydrate-associated metabolites showed directional differences between dietary groups, while nutrient digestibility and general health parameters remained largely unaffected. This study was conducted as an exploratory functional nutritional intervention integrating digestibility assessment, glycan-degrading enzyme activity, microbiome profiling, and serum metabolomics in a standardized canine feeding model. The findings should be interpreted as preliminary and hypothesis-generating rather than mechanistic proof of efficacy. Future studies involving larger cohorts, longer intervention periods, and expanded functional analyses will be necessary to validate these observations and to further investigate potential host–microbiome responses associated with dietary G. tenax supplementation in companion animals. Supplementary Materials The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ani16121786/s1, Table S1: Metadata and the OTU from 16S sequencing. Author Contributions Conceptualization, J.L.C.; data curation, W.Y.J. and S.C.; formal analysis, S.-Y.L., W.-D.L. and M.Y.L.; methodology, M.Y.L. and H.-W.C.; software, W.Y.J., W.-D.L. and H.-W.C.; validation, H.T.B., K.-M.S. and J.L.C.; investigation, W.Y.J., S.C., H.-W.C., M.Y.L., S.-Y.L., W.-D.L. and I.K.H.; writing—original draft, W.Y.J., S.C. and J.L.C.; writing—review and editing, W.Y.J., S.C., H.-W.C., M.Y.L., S.-Y.L., W.-D.L., H.T.B., K.-M.S. and J.L.C. All authors have read and agreed to the published version of the manuscript. Funding This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project No. RS-2023-00231446)”, Rural Development Administration, Republic of Korea. Institutional Review Board Statement The animal study protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the National Institute of Animal Science (NIAS) in Wanju, Republic of Korea (approval number NIAS2023-0615). (Approved 15 June 2023). Informed Consent Statement Not applicable. Data Availability Statement All data generated or analyzed during this study are included in this published article. Acknowledgments This study was supported by the 2026 RDA Fellowship Program of the National Institute of Animal Science, Rural Development Administration, Republic of Korea. Conflicts of Interest No potential conflicts of interest relevant to this article were reported. Abbreviations The following abbreviations are used in this manuscript: ADFI Average daily feed intake ASV Amplicon sequence variant ATTD Apparent total tract digestibility BCS Body condition score BH–FDR Benjamini–Hochberg false discovery rate BW Body weight BWG Body weight gain CA Crude ash CF Crude fiber CON Control group (diet containing 1% Ulva sp.) CP Crude protein CPMG Carr–Purcell–Meiboom–Gill DADA2 Divisive Amplicon Denoising Algorithm 2 DM Dry matter EE Ether extract GE Gross energy GT Gloiopeltis tenax supplementation group HPLC High-performance liquid chromatography ME Metabolizable energy MetaCyc Metabolic pathway database for microbial metabolism NFE Nitrogen-free extract NMR Nuclear magnetic resonance OPLS-DA Orthogonal partial least squares discriminant analysis PBS Phosphate-buffered saline PCA Principal component analysis PCoA Principal coordinates analysis PERMANOVA Permutational multivariate analysis of variance PICRUSt2 Phylogenetic Investigation of Communities by Reconstruction of Unobserved States 2 QIIME2 Quantitative Insights Into Microbial Ecology 2 RA Relative abundance SCFA Short-chain fatty acid TSP Trimethylsilyl propionate VIP Variable importance in projection References Figure 1. Principal coordinates analysis (PCoA) of fecal microbiota at week 4 based on ( A) Bray–Curtis, ( B) weighted UniFrac, and ( C) unweighted UniFrac distances. Distances were calculated from QIIME 2-derived ASV tables. Group differences were tested by PERMANOVA (999 permutations). CON, control diet; GT, Gloiopeltis tenax-supplemented diet (n = 5 per group). Figure 1. Principal coordinates analysis (PCoA) of fecal microbiota at week 4 based on ( A) Bray–Curtis, ( B) weighted UniFrac, and ( C) unweighted UniFrac distances. Distances were calculated from QIIME 2-derived ASV tables. Group differences were tested by PERMANOVA (999 permutations). CON, control diet; GT, Gloiopeltis tenax-supplemented diet (n = 5 per group). Figure 2. Phylum and family-level relative abundances of fecal microbiota on 4 weeks in dogs fed diets with or without G. tenax. Relative abundances at the phylum ( A) and family ( B) levels were calculated from ASV-based taxonomic profiles. CON, control; GT, G. tenax supplementation. Figure 2. Phylum and family-level relative abundances of fecal microbiota on 4 weeks in dogs fed diets with or without G. tenax. Relative abundances at the phylum ( A) and family ( B) levels were calculated from ASV-based taxonomic profiles. CON, control; GT, G. tenax supplementation. Figure 3. Predicted glycan and glycan metabolic pathways inferred by PICRUSt2 (MetaCyc database). The top 10 pathways ranked by mean relative abundance across both groups are shown. Bars represent mean ± SE for CON (gray, n = 5) and GT (orange, n = 5) groups. No pathways reached statistical significance (Wilcoxon rank-sum test, all p > 0.05). Figure 3. Predicted glycan and glycan metabolic pathways inferred by PICRUSt2 (MetaCyc database). The top 10 pathways ranked by mean relative abundance across both groups are shown. Bars represent mean ± SE for CON (gray, n = 5) and GT (orange, n = 5) groups. No pathways reached statistical significance (Wilcoxon rank-sum test, all p > 0.05). Table 1. Ingredient formulations and chemical compositions of experimental diets. Table 1. Ingredient formulations and chemical compositions of experimental diets. Component Experiment Diets CON GT Ingredient, % Rice flour 36.13 36.13 Chicken breast meal 14.08 14.08 Egg yolk powder 8.00 8.00 Lard 1.48 1.48 Cabbage powder 1.00 1.00 Green seaweed ( Ulva sp.) 1.00 - Seaweed tenax ( Gloiopeltis tenax) - 1.00 Tryptophan 0.01 0.01 Calcium carbonate 0.75 0.75 Monocalcium phosphate 0.95 0.95 Potassium citrate 1.00 1.00 Vitamin–mineral premix (1)0.40 0.40 Salt 0.20 0.20 Water 35.00 35.00 Chemical composition, DM % (analyzed) Crude protein 21.73 20.54 Ether extract 7.14 6.40 Crude fiber 0.31 0.20 Crude ash 4.01 3.75 Nitrogen-free extract 35.85 38.65 Gross energy, kcal/kg 3522.50 3500.00 (1) Vitamin and mineral premix supplied per kg of diets: 3500 IU vitamin A; 250 IU vitamin D 3; 25 mg vitamin E; 0.052 mg vitamin K; 2.8 mg vitamin B 1 (thiamine); 2.6 mg vitamin B 2 (riboflavin); 2 mg vitamin B 6 (pyridoxine); 0.014 mg vitamin B 12; 6 mg Cal-d-pantothenate; 30 mg niacin; 0.4 mg folic acid; 0.036 mg biotin; 1000 mg taurine; 44 mg FeSO 4; 3.8 mg MnSO 4; 50 mg ZnSO 4; 7.5 mg CuSO 4; 0.18 mg Na 2SeO 3; 0.9 mg Ca(IO 3) 2. Abbreviations: DM, dry matter. Table 2. Effects of G. tenax on feeding and body weight parameters in adult small-breed dogs. Table 2. Effects of G. tenax on feeding and body weight parameters in adult small-breed dogs. Parameter CON GT SE p-Value BW, kg Initial 4.14 4.08 0.368 0.908 1 w 4.29 4.23 0.405 0.919 2 w 4.33 4.28 0.426 0.938 Final 4.40 4.33 0.432 0.909 BCS Initial 3.60 3.60 0.332 1.000 1 w 4.00 4.20 0.469 0.771 2 w 4.00 4.20 0.412 0.740 Final 4.60 4.80 0.447 0.760 BWG, kg 0–1 w 0.15 0.15 0.041 0.973 1–2 w 0.04 0.05 0.029 0.777 2–4 w 0.07 0.05 0.031 0.600 ADFI, kg/d 0–1 w 133.60 132.60 9.017 0.939 1–2 w 137.20 136.20 9.780 0.944 2–4 w 137.20 136.20 9.780 0.944 Average fecal score 0–4 w 2.54 2.48 0.081 0.587 Abbreviations: CON, a basal diet containing 1% green seaweed ( Ulva sp.); GT, a basal diet without green seaweed and containing 1% seaweed tenax ( Gloiopeltis tenax); BW, body weight; BCS, body condition score; BWG, body weight gain; ADFI, average daily feed intake; SE, standard error. n = 5 per treatment. The CON group consisted of three Malteses (1F, 2M) and two Poodles (1F, 1M), and the GT group consisted of two Malteses (1F, 1M) and three Poodles (2F, 1M). F = female; M = male. Table 3. Effects of G. tenax on nutrient digestibility in adult small-breed dogs. Table 3. Effects of G. tenax on nutrient digestibility in adult small-breed dogs. Nutrient (%) CON GT SE p-Value DM 91.91 92.64 0.620 0.435 CP 95.88 96.38 0.292 0.267 EE 98.94 98.93 0.166 0.976 CF 54.99 50.78 6.169 0.647 CA 70.00 77.19 * 2.142 0.045 GE 97.44 97.59 0.246 0.671 NFE 98.36 98.67 0.382 0.587 Abbreviations: CON, a basal diet containing 1% green seaweed ( Ulva sp.); GT, a basal diet without green seaweed and containing 1% seaweed tenax ( Gloiopeltis tenax); DM, dry matter; CP, crude protein; EE, ether extract; CF, crude fiber; CA, crude ash; GE, gross energy; NFE, nitrogen-free extract; SE, standard error. * Significant difference between groups at p < 0.05 ( t-test). n = 5 per treatment. The CON group consisted of three Malteses (1F, 2M) and two Poodles (1F, 1M), and the GT group consisted of two Malteses (1F, 1M) and three Poodles (2F, 1M). F = female; M = male. Table 4. Effects of G. tenax on amino acid digestibility in adult small-breed dogs. Table 4. Effects of G. tenax on amino acid digestibility in adult small-breed dogs. Amino Acid (%) CON GT SE p-Value Threonine 97.26 96.89 0.330 0.451 Valine 97.51 97.21 0.293 0.486 Isoleucine 97.79 97.49 0.245 0.410 Leucine 98.00 97.71 0.235 0.410 Phenylalanine 97.67 97.39 0.267 0.484 Histidine 97.33 97.22 0.277 0.801 Lysine 97.22 96.79 0.328 0.378 Arginine 98.34 98.17 0.170 0.517 Methionine 97.26 96.87 0.355 0.461 Tryptophan 95.98 96.25 0.470 0.696 Aspartic acid 97.20 97.02 0.329 0.710 Serine 94.51 93.99 0.594 0.553 Glutamic acid 97.71 97.50 0.242 0.564 Proline 97.13 96.66 0.353 0.374 Glycine 96.82 96.47 0.369 0.517 Alanine 97.12 96.92 0.295 0.643 Tyrosine 97.25 96.59 0.364 0.239 Cystine 93.83 93.01 0.925 0.551 Abbreviations: CON, a basal diet containing 1% green seaweed ( Ulva sp.); GT, a basal diet without green seaweed and containing 1% seaweed tenax ( Gloiopeltis tenax); SE, standard error. n = 5 per treatment. The CON group consisted of three Malteses (1F, 2M) and two Poodles (1F, 1M), and the GT group consisted of two Malteses (1F, 1M) and three Poodles (2F, 1M). F = female; M = male. Table 5. Effects of G. tenax on glycan-degrading enzyme activity in adult small-breed dogs. Table 5. Effects of G. tenax on glycan-degrading enzyme activity in adult small-breed dogs. Enzyme (mM) CON GT SE p-Value Initial 4N-S 0.55 0.76 0.093 0.158 4N-FP 0.80 1.13 0.144 0.147 4N-NAG 0.97 0.81 0.137 0.426 4N-GalP 2.55 2.65 0.050 0.201 4N-GluP 2.30 2.34 0.018 0.124 Final 4N-S 0.59 1.47 * 0.115 <0.001 4N-FP 0.97 1.89 * 0.158 0.003 4N-NAG 0.98 * 0.61 0.101 0.035 4N-GalP 2.58 2.68 0.039 0.094 4N-GluP 2.35 2.40 0.028 0.300 Abbreviations: CON, a basal diet containing 1% green seaweed ( Ulva sp.); GT, a basal diet without green seaweed and containing 1% seaweed tenax ( Gloiopeltis tenax); 4N-S, 4-nitrophenyl-sulfatase; 4N-FP, 4-nitrophenyl-fucosidase; 4N-NAG, 4-nitrophenyl-N-acetyl- β-glucosaminidase; 4N-GalP, 4-nitrophenyl-galactosidase; 4N-GluP, 4-nitrophenyl-glucosidase; SE, standard error. * Significant difference between groups at p < 0.05 ( t-test). n = 5 per treatment. The CON group consisted of three Malteses (1F, 2M) and two Poodles (1F, 1M), and the GT group consisted of two Malteses (1F, 1M) and three Poodles (2F, 1M). F = female; M = male. Table 6. Fecal microbial alpha-diversity indices in dogs fed diets with or without G. tenax. Table 6. Fecal microbial alpha-diversity indices in dogs fed diets with or without G. tenax. Index CON GT p-Value (Mean ± SD) (Mean ± SD) Chao1 ୮୧.୮୦ ବ୍ଦ ୧୦.୪୭ ୮୦.୦୦ ବ୍ଦ ୧୪.୮୮ 0.831 Shannon Entropy ୩.୧୩ ବ୍ଦ ୦.୨୪ ୩.୧୩ ବ୍ଦ ୦.୩୪ 0.963 Simpson ୦.୯୧ ବ୍ଦ ୦.୦୩ ୦.୯୨ ବ୍ଦ ୦.୦୪ 0.76 Observed Features ୮୧.୮୦ ବ୍ଦ ୧୦.୪୭ ୮୦.୦୦ ବ୍ଦ ୧୪.୮୮ 0.831 Evenness ୦.୭୧ ବ୍ଦ ୦.୦୪ ୦.୭୨ ବ୍ଦ ୦.୦୬ 0.854 Values are presented as mean ± SD (n = 5 per group). p-values were obtained using the Wilcoxon rank-sum test (two-sided). CON, control; GT, G. tenax supplementation. Table 7. Serum metabolite changes between CON and GT. Table 7. Serum metabolite changes between CON and GT. Tier Metabolite CON Δ (After-Before) GT Δ (After-Before) ΔΔ (GT−CON) p-Value (Δ Group) FDR Significant Glutamine −7.63 2.68 10.30 0.0002 0.004 * Significant Methionine −0.66 0.48 1.13 0.004 0.047 * Trend Alanine −2.37 2.45 4.82 0.022 0.160 Trend Serine 0.96 −1.54 −2.50 0.031 0.170 Trend Histidine −1.06 0.45 1.51 0.066 0.290 Trend Pyruvate −0.35 0.85 1.19 0.083 0.293 Trend Creatine 0.02 0.85 0.83 0.093 0.293 Not significant Lactate 4.47 −1.41 −5.88 0.139 0.383 Not significant Trimethylamine N-oxide 0.11 0.16 0.05 0.234 0.532 Not significant Proline 0.54 2.90 2.36 0.266 0.532 Not significant Leucine −0.43 0.91 1.35 0.283 0.532 Not significant Isoleucine 0.02 0.46 0.44 0.290 0.532 Not significant Valine 0.38 1.64 1.26 0.316 0.535 Not significant Threonine −0.65 2.15 2.79 0.408 0.641 Not significant Acetone 0.01 0.14 0.13 0.589 0.860 Not significant Dimethylsulfone −0.14 −0.14 −0.01 0.626 0.860 Not significant Glucose −42.26 −46.26 −4.00 0.759 0.982 Not significant Glycerol −2.23 −0.40 1.82 0.898 0.989 Not significant Methanol −1.01 −1.53 −0.51 0.912 0.989 Not significant Acetoacetate 0.00 −0.12 −0.11 0.933 0.989 Not significant Betaine 1.32 1.49 0.17 0.959 0.989 Not significant Glycine −3.65 −1.55 2.10 0.989 0.989 Δ values calculated as 4 weeks − day 0 for each metabolite. CON, control; GT, G. tenax supplementation. * Significant difference between groups at q < 0.05. Table 8. Variable importance in projection (VIP) scores from OPLS-DA based on serum Δ metabolite profiles (Δ = 4 weeks − day 0). Table 8. Variable importance in projection (VIP) scores from OPLS-DA based on serum Δ metabolite profiles (Δ = 4 weeks − day 0). Rank CON (0–4 Weeks) VIP GT (0–4 Weeks) VIP 1 Glycine (2.01) Creatine (1.48) 2 Glutamine (1.31) Serine (1.45) 3 Serine (1.27) Glucose (1.32) 4 Valine (1.26) Glycine (1.24) 5 Methionine (1.24) Methionine (1.22) Variables were ranked by variable importance in projection (VIPpred/VIP) derived from OPLS-DA models built on Pareto-scaled data in SIMCA-P+ (ver. 12.0). CON, control; GT, G. tenax supplementation. Table 9. Spearman rank correlations between week 4 fecal microbiome family-level relative abundances and serum Δ metabolite values (Δ = 4 weeks − day 0). Table 9. Spearman rank correlations between week 4 fecal microbiome family-level relative abundances and serum Δ metabolite values (Δ = 4 weeks − day 0). Microbial Family Metabolite(s) Correlation ( r) Direction Prevotellaceae Alanine, Methionine 0.62 Positive Lachnospiraceae Glutamine 0.55 Positive Sutterellaceae Creatine 0.48 Positive Fusobacteriaceae Serine –0.44 Negative Correlation coefficients ( r) indicate the direction and strength of monotonic associations between microbial families and intervention-associated changes in serum metabolites. Correlation p-values were adjusted for multiple testing using the Benjamini–Hochberg false discovery rate (BH-FDR) procedure (i.e., correction applied to correlation p-values). These correlations are presented as exploratory, associative evidence and do not imply causal relationships. 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Dietary Gloiopeltis tenax Is Associated with Shifts in Fecal Microbiome and Serum Metabolome Profiles in Healthy Adult Dogs. Animals. 2026; 16(12):1786. https://doi.org/10.3390/ani16121786 Chicago/Turabian Style Jung, Won Yong, Seyeon Chang, Han Tae Bang, Kyoung-Min So, Min Young Lee, Sang-Yeob Lee, Woo-Do Lee, Hyun-Woo Cho, Il Ki Hwang, and Ju Lan Chun. 2026. "Dietary Gloiopeltis tenax Is Associated with Shifts in Fecal Microbiome and Serum Metabolome Profiles in Healthy Adult Dogs" Animals 16, no. 12: 1786. https://doi.org/10.3390/ani16121786 APA Style Jung, W. Y., Chang, S., Bang, H. T., So, K.-M., Lee, M. Y., Lee, S.-Y., Lee, W.-D., Cho, H.-W., Hwang, I. K., & Chun, J. L. (2026). Dietary Gloiopeltis tenax Is Associated with Shifts in Fecal Microbiome and Serum Metabolome Profiles in Healthy Adult Dogs. Animals, 16(12), 1786. https://doi.org/10.3390/ani16121786
