Supplementation of Yeast Cell Wall Fraction Tends to Improve Intestinal Health in Adult Dogs Undergoing an Abrupt Diet Transition

When owners decide to change their pet's food, a rapid transition may cause gastrointestinal distress. Yeast products may help with digestive upset during diet transition due to the bioactive compounds they possess, which may lead to improved intestinal morphology and integrity, modified gut microbiota, and modulated immune responses. The objective of this study was to determine the effects of a yeast cell wall fraction supplement on measures of gut integrity and fecal characteristics of adult dogs undergoing an abrupt diet transition. Twelve adult female beagles (mean age: 5.16 ± 0.87 years; mean body weight: 13.37 ± 0.68 kg) were used in a replicated 4 × 4 Latin square design with four 28-day experimental periods. During days 1–14, dogs were fed a dry kibble diet and supplemented with a placebo (cellulose; 125 mg/d) or yeast product (365 mg/d; equivalent to 0.2% of diet). During days 15–28, dogs remained on their placebo or yeast treatments but were rapidly transitioned to a canned diet or high-fiber diet. Fresh fecal samples were collected on days 13, 16, 20, 24, and 27 for measurement of pH, dry matter, calprotectin, immunoglobulin A (IgA), Escherichia coli, and Clostridium perfringens. Blood samples were collected on days 14, 17, and 28 to measure serum lipopolysaccharide-binding protein concentrations. All data were analyzed using the Mixed Models procedure of SAS 9.4. Fecal pH, dry matter, calprotectin, IgA, and E. coli were not affected (P > 0.05) by treatment before diet transition. Dogs supplemented with yeast cell wall fraction tended to have higher (P = 0.06) fecal C. perfringens counts than the controls. After diet transition, most parameters were not altered (P > 0.05) by treatment except that yeast-supplemented dogs tended to have higher (P = 0.06) fecal IgA than controls. Our results suggest that the yeast product may modestly improve intestinal health after an abrupt diet transition in adult dogs by enhancing intestinal immunity.


INTRODUCTION
Diet transition commonly occurs in dogs when owners purchase new products. If not transitioned slowly, digestive upset may occur, which results in poor stool quality and gastrointestinal (GI) upset. Based on past research, the supplementation of functional ingredients such as yeast products may serve as a potential approach to minimizing digestive upset in such situations.
Yeast products are ingredients containing yeast cells or yeast derivatives and are commonly used in livestock feeds to improve animal performance (1,2). Yeast products have also been shown to improve GI health in various ways. For example, dogs (3)(4)(5)(6) and cats (7) supplemented with yeast products had reduced potential pathogenic bacteria (e.g., Escherichia coli; Clostridium perfringens) and increased beneficial bacteria (e.g., Lactobacillus; Bifidobacterium) in feces. In pigs (2,8) and chickens (1), supplementation of yeast products has led to increased small intestinal villus height and/or villus height: crypt depth ratio. Yeast supplementation enhanced colonic barrier function and decreased intestinal permeability in murine intestinal obstruction and colitis models (9,10). Lastly, yeast-supplemented dogs (4,11) and pigs (12) had greater small intestinal immunoglobulin A (IgA) concentrations vs. controls.
Results from those studies indicate that supplementation of yeast products may improve GI health by modifying gut microbiota, improving intestinal morphology and integrity, and/or modulating immune responses. In this study, we aimed to determine the effects of a yeast cell wall fraction product on measures of GI tract stability and fecal characteristics of adult dogs undergoing an abrupt diet transition. We hypothesized that yeast cell wall fraction-supplemented dogs would have greater gastrointestinal stability and protection from a disrupted gut barrier function due to abrupt diet change, as well as reduced fecal pathogens compared to control dogs receiving a placebo treatment.

Animals, Diets, and Treatments
All animal care and experimental procedures were approved by the University of Illinois Institutional Animal Care and Use Committee (Protocol No. 17276) prior to experimentation. All methods were performed in accordance with the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals. Twelve adult female beagles (mean age: 5.16 ± 0.87 years; mean BW: 13.37 ± 0.68 kg) were used and housed individually in pens (1.0 m wide × 1.8 m long) in a humidityand temperature-controlled room on a 14 h light: 10 h dark cycle. Dogs had access to fresh water ad libitum at all times. All diets were offered to dogs once a day in the morning to maintain body weight throughout the study. Dogs were weighed and body condition scores were assessed using a 9-point scale (13) weekly before feeding.
A replicated 4 × 4 Latin square design was conducted. Each 28-d experimental period consisted of an adaptation phase (d 1-14) and a diet transition phase (d [15][16][17][18][19][20][21][22][23][24][25][26][27][28]. Three diets that met all Association of American Feed Control Officials (AAFCO) (14)  The yeast cell wall fraction tested was from Saccharomyces cerevisiae and the analyzed chemical composition is presented in Table 3. During the adaptation phase, all dogs were fed the experimental diet and supplemented with placebo or yeast product. During the diet transition phase, dogs remained on their placebo or yeast treatments, but were fed their new diet (CD or HFD). Therefore, dogs were allotted to one of four groups each experimental period: (1) Yeast CD; (2) Yeast HFD; (3) Placebo CD; and (4) Placebo HFD.

Fecal Sample Collection
On d 13, 16, 20, 24, and 27 of each experimental period, one fresh fecal sample from each dog was collected within 15 min of defecation. Fecal samples were scored according to a 5point scale: 1 = hard, dry pellets, small hard mass; 2 = hard, formed, dry stool; remains firm and soft; 3 = soft, formed, and moist stool, retains shape; 4 = soft, unformed stool, assumes shape of container; and 5 = watery, liquid that can be poured. Fecal pH was measured immediately using an AP10 pH meter (Denver Instrument, Bohemia, NY) equipped with a Beckman Electrode (Beckman Instruments Inc., Fullerton, CA). An aliquot of fresh feces was dried at 105 • C for 2 d for dry matter (DM) determination. On d 13, 20, and 27 of each experimental period, two more aliquots of fresh fecal samples were collected for determination of fecal IgA, calprotectin and potential pathogenic bacteria (E. coli and C. perfringens). For fecal IgA and calprotectin measurements, the aliquot was stored at −80 • C until analyses. For fecal bacteria analyses, the aliquot was processed for bacterial culture immediately after collection.

Food Intake and Body Weight
Average daily food, energy, and nutrient intakes, including protein, fat, TDF, and NFE were not changed by yeast treatment prior to diet transition ( Table 4). Post-transition diets affected nutrient intakes. Daily intakes of protein, fat, and energy were higher (P < 0.01), while daily intakes of TDF and NFE were lower (P < 0.01) in dogs fed the CD than those fed the HFD ( Table 5).
Average BW (13.52 kg) and body condition score (7.15/9) were not altered by yeast treatment prior to and after diet transition ( Table 4; Figure 1A). Body weight was affected by diet after diet transition. Dogs consuming CD had greater decreases (P < 0.01) in BW than dogs consuming HFD ( Figure 1A).

Fecal Characteristics
Fecal characteristics, including pH, score, and DM were not altered by yeast treatment prior to diet transition ( Table 6). After diet transition, the change from baseline values for fecal characteristics were affected by diet and time, but not yeast treatment (Figure 1). Fecal pH decreased after diet transition. The change from baseline values for fecal pH were greater (P < 0.01) in dogs fed HFD than in those consuming CD. The decrease in pH on d 16 was larger (P < 0.01) than other days ( Figure 1B). Fecal scores increased (wetter feces) after diet transition. The increases in fecal scores were greater (P < 0.01) in dogs fed HFD vs. dogs fed CD, with the largest change from baseline appearing on d 20 (P < 0.01) (Figure 1C). Fecal DM decreased after diet transition ( Figure 1D). Dogs consuming CD had larger decreases (P = 0.03) in fecal DM than dogs consuming HFD.

Fecal Bacteria, IgA, and Calprotectin
Fecal E. coli, IgA, and calprotectin were not affected by yeast treatment prior to diet transition ( Table 6). Dogs supplemented with yeast product tended to have higher (P = 0.06) fecal C. perfringens counts than control dogs. After diet transition, fecal bacteria were changed by diet, but not yeast treatment. Fecal E. coli decreased in HFD-fed dogs but increased in CD-fed dogs (P < 0.05; Figure 1E). Fecal C. perfringens increased in dogs fed HFD, but decreased in dogs fed CD (P < 0.01; Figure 1F). The change in fecal IgA after diet transition was affected by treatment and diet. Yeast-supplemented dogs tended to have higher (P = 0.06) fecal IgA than the control dogs. In addition, fecal IgA increased (P < 0.01) in HFD-fed dogs, but decreased (P < 0.01) in CD-fed dogs ( Figure 1G). Finally, fecal calprotectin increased after diet transition ( Figure 1H). Dogs consuming CD tended to produce more (P = 0.06) calprotectin than dogs consuming HFD.

Serum LPS-Binding Protein
Serum LPS-binding protein concentration was not affected by yeast treatment prior to diet transition ( Table 6). After diet transition, serum LPS-binding protein increased, but the changes from baseline were not affected by treatment or diet ( Figure 1I).

DISCUSSION
Diet transition occurs when pet owners purchase different products, which might result in GI discomfort. Yeast products may serve as a functional ingredient in dog foods and may protect animals from digestive upset due to its benefits on GI health. Here, we investigated the effects of yeast cell wall fraction on fecal characteristics and GI tract stability in adult dogs undergoing diet transition from a dry kibble diet to a high-fiber diet or canned diet. Our findings revealed that before diet transition, all indices except for fecal C. perfringens counts, were not altered by yeast supplementation. After diet transition, all measurements, except for fecal IgA concentration were not changed by yeast treatment. Fecal IgA concentrations tended to increase in yeast-supplemented dogs. Diet transition led to significant changes to fecal characteristics and inflammatory biomarkers. Fecal pH decreased after dogs were fed HFD, which was expected. Lower fecal pH is due primarily to greater short-chain fatty acid concentrations as a result of fiber fermentation by microbiota in the hindgut (21,22). Fecal scores increased and fecal DM decreased when dogs were fed both HFD and CD, indicating wetter and/or looser stools. Fecal calprotectin, a protein complex present in neutrophils, monocytes and reactive macrophages, has been reported to be increased in dogs with chronic diarrhea and chronic inflammatory enteropathies (23,24). Here, we observed increased fecal calprotectin concentrations after diet transition, which reveals a slight elevation in GI inflammation. Finally, serum LPS-binding protein concentrations were elevated after diet transition. LPS binding-protein binds to endotoxins, including LPS from Gram-negative bacteria, with their concentration being increased after endotoxemia or bacteremia (25,26). The increased serum LPS-binding protein concentrations in this study suggested greater GI permeability (leaky gut) after diet transition.
Fecal IgA was the only parameter that tended to be affected by yeast supplementation after diet transition. Elevated secretory IgA concentrations indicate enhanced mucosal immunity. Secretory IgA in the intestine plays a role in protecting mucosal surface against enteric antigens by inhibiting colonization and invasion of pathogens as well as food antigen uptake (27,28). This response was in agreement with previous studies that reported elevated IgA concentrations in dogs consuming yeast products, indicating enhanced mucosal immunity. Middelbos et al. (4) noted that ileal IgA concentrations tended to respond quadratically (P = 0.09) to yeast cell wall supplementation in adult healthy dogs, with the highest concentration coming from the 0.25% supplementation level. Swanson et al. (11) supplemented 0.25% mannanoligosaccharides (MOS), the main component of the yeast cell walls, to adult healthy dogs and observed a tendency (P = 0.06) of increased ileal IgA concentrations. Increased IgA concentration may possibly be explained by two major components of the yeast cell walls, beta-glucans and MOS. Beta-glucans have been shown to be an immunostimulant that modulates innate and adaptive immune responses in various animal species, including dogs (29)(30)(31). As an immunostimulant, beta-glucans induce a cascade of immune responses by binding to dectin-1 receptors that are expressed on immune cells such as macrophages, monocytes, dendritic cells and neutrophils (32). The possible mechanisms by which MOS induce immune responses include the binding of mannose receptors that are expressed on antigen-presenting cells, including macrophage and dendritic cells (33). This response is supported by findings from previous studies in dogs, broilers, and pigs whereby oral administration of MOS led to enhanced immunity (34)(35)(36)(37).
Supplementation of yeast products have been shown to decrease potential pathogenic bacteria such as E. coli in previous studies where dogs were supplemented with yeast cell wall, MOS or live S. cerevisiae (4,5,37). Decreased pathogenic bacteria are believed to be due to the ability of yeast cell wall to bind and inactivate bacteria. Mannose in yeast cell walls binds to type-1 fimbriae of pathogenic bacteria and thus prevent their adhesion and colonization to the host mucosa (38). We did not observe changes to fecal E. coli in this study, however, which is in line with findings from Swanson et al. (11) where dogs were supplemented with MOS. Although decreased fecal C. perfringens was noted in cats consuming yeast cell walls (7), lower fecal C. perfringens was not observed in dogs supplemented with MOS or live S. cerevisiae (5,11,37). Middelbos et al. (4) reported a cubic response of fecal C. perfringens counts to yeast cell wall supplementation in dogs, with greatest counts being observed at the 0.25% supplementation level. This is similar to our findings in dogs supplemented with 0.2% yeast cell wall fractions, who tended to have greater fecal C. perfringens counts compared to the control dogs before diet transition. Fecal C. perfringens counts in our study were 8.0-8.4 colonyforming units (CFU) log 10 /g DM, which is lower than those (9.5-10.0 CFU log 10 /g DM) reported by Middelbos et al. (4). The difference could be contributed by several factors, including diet composition, dog breed studied, and plating methods used to quantify microbes. The increased fecal C. perfringens, however, did not negatively affect measures of GI distress. C. perfringens are parts of commensal microbiota in dogs and can be cultured from more than 80% of healthy dogs and diarrheic dogs (39,40). Therefore, greater fecal C. perfringens counts usually do not lead to diarrhea. Canine C. perfringens-associated diarrhea is more likely the result of intestinal microbiota disruption, which enables sporulation of commensal C. perfringens and production of enterotoxin (41). It also has been shown that C. perfringens enterotoxin is only detected in 5-15% of healthy dogs but in 34% of diarrheic dogs (39,40). Fecal enterotoxin concentrations were not evaluated in the current study, but was unlikely because fecal scores were not affected by yeast cell walls.
The lack of significant effects on most intestinal health indices by yeast supplementation in this study could be due to the short treatment period used or the low supplement dosage tested. Grieshop et al. (37) observed decreased fecal E. coli counts in dogs supplemented with 1% MOS for 28 d. In dogs with enteropathogenic E. coli-induced diarrhea, supplementation of 2 g/kg BW MOS for 20 d resulted in faster recovery (42). Therefore, testing the yeast cell wall fractions with longer supplementation periods and/or higher dosages may be suggested in the future.
In conclusion, supplementation of 0.2% yeast cell wall fractions to dogs tended to increase fecal IgA concentrations, but did not affect fecal characteristics, fecal bacterial populations, or serum LPS-binding protein concentrations after an abrupt diet transition. Inclusion of yeast cell wall fractions in diets may modestly improve intestinal health for dogs undergoing diet transition.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The animal study was reviewed and approved by the University of Illinois Institutional Animal Care and Use Committee.

AUTHOR CONTRIBUTIONS
KS and RR designed the experiment. C-YL and MM performed all laboratory analyses. C-YL and MC performed the animal trial and sample collection. C-YL conducted statistical analysis and wrote the manuscript. All authors have read and approved the manuscript.

FUNDING
The authors declare that this study received funding from Phileo by Lesaffre. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.