Functional properties of Ganoderma lucidum supplementation in canine nutrition

Abstract

Ganoderma lucidum (GL) is a mushroom that has been widely used in Asia for its immunostimulatory and anti-inflammatory capacity, which has been hypothesized to be attributed mainly to the recognition of its cell-surface patterns by cells of the immune system present in the gastrointestinal tract, resulting in a cascade of modulatory events. However, the nutraceutical properties of GL have not been tested in dogs. Forty adult beagles were used in a completely randomized design. The objective of the present study was to evaluate the effects of dietary inclusion of GL on peripheral blood mononuclear cells (PBMC; T cells, B cells, monocytes, and natural killers), vaccine response, nutrient digestibility, fecal fermentative end-products, and skin and coat quality of adult dogs. Dogs were fed a commercial dry extruded complete and balanced diet plus GL top-dressed daily upon feeding time. Four experimental treatments were used: 0% GL supplementation (control), 5 mg/kg BW of GL, 10 mg/kg BW of GL, or 15 mg/kg BW of GL. Following a 7 d adaptation to the control diet, dogs were fed their respective treatment diets for 28 d. They were challenged with vaccination of a modified live virus Canine Distemper, Adenovirus Type 1 (Hepatitis), Adenovirus Type 2, Parainfluenza, and Parvovirus and killed Rabies Virus on day 7 with blood collections on days 0, 14, and 28. The inclusion of GL in all dosages was well-accepted by all dogs, with no detrimental effect on macronutrient apparent total tract digestibility. There was a trend that the percentage of major histocompatibility II (MHC-II) from B cells was greater in dogs fed 15 mg/kg of GL (41.91%) compared to the control group (34.63%). The phagocytosis response tended to have treatment-by-time interaction among treatments; dogs fed 15 mg/kg of GL tended to have greater phagocytosis activity on day 28 than dogs from the control group and dogs fed 5 mg/kg of GL. The vaccine-specific serum immunoglobulin G (IgG) concentrations were higher in the group supplemented with 15 mg/kg of GL compared to treatment control 7 d after the vaccination for rabies. These data suggest that the inclusion of GL had no detrimental effects on any analyzed PBMC. Due to changes in immune parameters among treatments, GL may also exert beneficial immunostimulatory effects in healthy adult dogs when provided at a daily dose of 15 mg/ kg BW.

Ganoderma lucidum is a novel functional ingredient in canine nutrition. Results found that it may exert beneficial immunostimulatory effects in healthy adult dogs.

Introduction

Ganoderma lucidum (GL) is a mushroom, also known as Reishi or Lingzhi, of glossy exterior and woody texture that has been widely used in Asia for its medicinal properties (Wu et al., 2016). Some of the health benefits related to the consumption of GL include immunostimulatory, anti-inflammatory, anti-viral, antioxidant, and antitumor properties (Yang et al., 2010; Barbieri et al., 2017). Among the bioactive compounds present in mushrooms, polysaccharides, particularly β-glucans have been attributed to their nutraceutical properties (Wu et al., 2016). β-glucans are present in the cell wall of mycetes and are comprised of linear β-1,3/1,6 glucans, with β-1,3 glucan as the main active compound (Kavishree et al., 2008). The content and biological activity of β-glucans in mushrooms depend on many factors such as species, growing conditions, degree of fruiting body maturity, dietary fiber content, molecular weight, conformation, frequency of bonds, solubility, and changes in structure (Kyanko et al., 2013).

The immunostimulatory activity of β-glucan occurs as a result of its ability to behave similarly to pathogen-associated molecular patterns and be recognized by and attach to specific cell-surface pattern-recognition receptors present on the immune cells (e.g., Toll-like receptors [TLR] and NOD-like receptors), which in turn stimulates the immune system, activating macrophage recruitment and promoting phagocytic activity (Wu et al., 2016). Studies on mice revealed that GL polysaccharides have the potential to modulate the composition of the gut microbiota and have prebiotic-like effects, improving gut barrier integrity, reducing endotoxemia, decreasing TLR4 signaling, and decreasing inflammation (Chang et al., 2015; Chen et al., 2020).

Studies about the effects of β-glucan supplementation on dogs focus mostly on yeast sources. Stercova et al. (2016) showed a beneficial modulation of the gut microbiota composition with fewer Escherichia coli and fecal enterococci counts in the feces of dogs consuming 2.9 × 10 cfu/kg BW of a live yeast strain. De Souza Theodoro et al. (2019) reported a reduction of the systemic inflammatory activity of dogs, with lower interleukin six in dogs’ serum, consuming a diet containing 0.3% yeast extract. However, the potential nutraceutical effects of GL, a fungi β-glucan source, have not been investigated in dogs. Thus, this study aimed to determine the dose–response effects of GL on apparent total tract digestibility (ATTD) of macronutrients, fecal metabolites and microbiota, immunological parameters, and skin and coat health in adult dogs.

Materials and Methods

The Kennelwood, Inc. Animal Care and Use Committee approved all animal care procedures before animal experimentation. All methods were performed in accordance with the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals.

Animals and dietary treatments

Forty adult beagles (mean age = 9.1 ± 2.76 yr; mean BW = 15.5 ± 3.91 kg) were used in a completely randomized design. The dogs were housed individually in pens with nose-to-nose contact with dogs in adjacent runs and visual contact with all dogs in a temperature-controlled room at Kennelwood Inc., Champaign, IL. All animals were fed once daily at 1700 hours with unlimited access to water. Animals were fed to maintain body weight, while intake and refusals were recorded after each meal. Dogs were fed a commercial dry extruded complete and balanced diet (Purina Dog Chow Complete Adult Dry Food with Real Chicken, Purina, St. Louis, MO). Daily supplementation of GL drum dried (Sourced from Alura Inc., Durham, NA 27703; GL composition provided by sourcing company: β-glucans ≥ 70%, ashes < 3%, total fat < 2%, protein < 4%, humidity < 8%) was added to 15 g of commercial wet pet food (Purina Dog Chow High Protein Classic Ground Wet Dog Food with Chicken) top-dressed daily upon feeding time. Dogs were randomized to one of the four experimental treatments: 0% GL supplementation (control), 5 mg/kg BW of GL, 10 mg/kg BW of GL, or 15 mg/kg BW of GL.

Weekly body weight and body condition score assessments were taken during the morning period. Animal body condition score was assessed on a 9-point scale, with ‘1’ being severely malnourished, ‘5’ being an ideal body weight, and ‘9’ being severely obese (Laflamme, 1997).

Experimental design and sample collection

The study began with a 7 d adaptation period to the control diet. After the adaption period, dogs were randomly assigned to one of the four experimental diets. Dogs were fed their assigned diets for a total of 28 d. On day 7, all animals were vaccinated with a modified live virus Canine Distemper, Adenovirus Type 1 (Hepatitis), Adenovirus Type 2, Parainfluenza, and Parvovirus (Duramune Max 5, Elanco), and with a killed Rabies Virus (Rabvac 3, Boehringer Ingelheim). On days 0, 14, and 28, blood samples were collected from the jugular vein for analyses of serum chemistry and complete blood count, and determination of immune cell number, phagocytic capacity, and vaccine-specific antibody response. Skin and coat scores were performed on days 0 and 28 according to the methods described below. Additionally, at the end of the experimental period, a 4-d total fecal collection was performed to determine macronutrient digestibility and fecal scores of dogs fed assigned treatments.

At the baseline and during the 4-d of fecal collection phase at the end of the experimental phase, a fresh fecal sample was collected from each dog within 15 min of defecation for dry matter (DM) and fecal metabolite analysis. The pH, as-is weight, and fecal score were measured for each sample. The fresh samples were aliquoted for DM, phenols and indoles, short-chain fatty acids (SCFA), branched-chain fatty acids (BCFA), and ammonia determination and stored at − 20 °C until later analysis. A separate fecal aliquot was placed into sterile cryovials and stored at − 80 °C for analysis of fecal microbiota, fecal immunoglobulin A (IgA), and calprotectin.

A fasted blood sample was collected via jugular venipuncture and immediately placed into respective collection tubes. Two 10 mL aliquots were placed into K2EDTA tubes (BD Vacutainer, K2 EDTA 18 mg, Franklin Lakes, NJ) to be used for immunoassays. A 5 mL aliquot was placed into a Serum Separator Tube (BD Vacutainer, SST, Franklin Lakes, NJ) to be used for serum chemistry and immunoglobulin concentrations. A 1 mL aliquot was placed into a K2EDTA tube (BD Vacutainer, K2EDTA 18 and 3.6 mg, Franklin Lakes, NJ) to be used for a complete blood count. The 5 mL blood aliquot placed into the Serum Separator Tube (BD Vacutainer, SST, Franklin Lakes, NJ) was centrifuged (1,300 × g at 4 °C), and supernatants were pipetted into cryovials and were immediately frozen at −80 °C and stored until further vaccine-response immunoglobulin analysis. A 2 mL aliquot was placed into a K2EDTA tube (BD Vacutainer, K2 EDTA 18 and 3.6 mg, Franklin Lakes, NJ) to be used for phagocytic capacity analysis.

Immune cell populations

Peripheral blood mononuclear cells (PBMC) were analyzed for major lymphocyte populations natural killer (NK); helper-T (Th), cytotoxic-T (Tc), and their interferon-gamma- (IFN-γ) producing subsets Th1 and Tc1, respectively, as well as two classical antigen-presenting (AP) cells (i.e., monocytes and B cells). Histopaque-1,077 (Sigma-Aldrich, St. Louis, MO) was added to whole blood in a 1:1 volume ratio and centrifuged at 400 × g at 25 °C for 30 min on the lowest acceleration and with the brake off to separate PBMC from blood. The percentage of T-cells and AP cells was evaluated by flow cytometry. For T-cell populations, PBMC were distributed into two tubes (1 × 10⁶ cell/tube) with one tube as control (non-stimulated) and the other stimulated with a cell stimulation cocktail (phorbol 12-myristate 13-acetate, ionomycin, brefeldin A, and monensin; eBioscience, San Diego, CA). Both tubes were incubated at 37 °C in 5% CO₂ for 4 h followed by surface marker labeling with antibodies including anti-CD3-fluorescein isothiocyanate (FITC), anti-CD4-allophycocyanin (APC), anti-CD8-pacific blue (Bio-Rad Laboratories, Inc., Hercules, CA) and viability dye (eFluor 780; eBioscience). After labeling, cells were fixed and permeabilized for 20 min with fixation buffer and permeabilization buffer (eBioscience). Intracellular marker staining was then performed with anti-IFN-γ-phycoerythrin (PE; Bio-Rad Laboratories, Inc.). The AP cells of interest include B cells and monocytes presenting major histocompatibility complex class II (MHC-II) on the cell-surface. One aliquot of PBMC (1 × 10⁶ cells/tube) was stained with anti-CD14-APC, anti-CD21-PE, anti-MHC-II-FITC antibodies (Bio-Rad Laboratories, Inc.), and viability dye (eFluor 780; eBioscience). Populations of T-cells and AP cells were then determined by a BD LSR flow cytometer (Becton Dickinson, Franklin Lakes, NJ). Mean fluorescence intensity was calculated based on the geometric mean of MHC-II-FITC fluorescence intensity values. Flow cytometry data were analyzed using FCS Express 5 Flow Cytometry Software (De Novo Software, Glendale, CA).

Responsiveness of leukocytes to TLR agonists

PBMC (25 × 10⁵ cells/well) were seeded into 96-well plates. Control wells had no agonist added, while an agonist of TLR2 (10 µg/mL Zymosan; Invivogen, San Diego, CA), TLR3 (50 µg/mL polyinosinic-polycytidylic acid sodium salt, poly [I: C]; Sigma, St. Louis, MO), TLR4 (100 ng/mL LPS; Sigma), and TLR 7/8 (5 µg/mL resiquimod, Invivogen, San Diego, CA) were added into respective wells. Cells then were incubated at 37 °C in 5% CO₂ for 24 h, centrifuged at 300 × g with full acceleration and full brake for 10 min at 4 °C, and supernatants were transferred to 96-well plates (Thermo Fisher Scientific, Waltham, MA) and stored at −80 °C until time of analysis. Frozen samples were thawed and composited for measurements of tumor necrosis factor-alpha (TNF-α) concentration using a commercial Elisa kit (R&D Systems, Minneapolis, MN) in duplicate, and mean values were expressed as pg/mL of cell supernatant.

Phagocytosis capacity

Blood samples were collected via jugular venipuncture into EDTA tubes (2 mL). All blood samples began being stained and further processed within 1 h of collection. 20 µL of primary AlexaFlour 647-conjugated mouse anti-human CD14 antibody (Clone TUK4, Product Code MCA1568A647, Bio-Rad) was added to 500 µL aliquots of whole blood and incubated for 30 min at 4 °C in the dark. Red blood cells were then lysed using 10× BD Pharmlyse™ red cell lysis buffer (catalog #555899, BD, Franklin Lakes, NJ USA) as per company instructions. Samples were then centrifuged at 400 × g for 5 min. The supernatant was discarded, and the sample was then washed twice with a cold, sterile PBS buffer. PBMC were then stained using anti-PE MicroBeads UltraPure (order # 130-105-639, MACS, Miltenyi Biotec) and passed through an MS column (order # 130-042-201, MACS, Miltenyi Biotec) as per company instructions. Phagocytosis assays were performed using black flat-bottom 96-well tissue culture plates (Corning, Los Angeles, CA). All the incubations were done in a humidified 37 °C and 5% CO₂ incubator. Macrophages retained in the column separation were resuspended in RPMI supplemented with 7.5% FBS. Cell concentration was adjusted to 1 × 10⁶/mL, and 100 µL of cell suspension was pipetted into each well. The plate was incubated for 2 h to allow cells to adhere to the bottom of the wells. Then, the culture medium was aspirated. The E. coli suspension was briefly sonicated to disperse any aggregates, and 100 µL were added to each well. The plate was covered and incubated for 20 h. After incubation, the buffer in the plate was removed by aspiration. Extracellular fluorescence was quenched by adding 100 µL of trypan blue (250/zg/mL, pH 4.4). Wells containing only fluorescent particles were used as controls to indicate complete quenching. After 1 min, the dye was removed. The fluorescence intensity (relative fluorescence unit, RFU) was determined at 485 + 10 nm excitation and 530 ± 12.5 nm emission wavelengths using an automated fluorescence microplate reader.

The phagocytosis response to the treatments was calculated as follows:

$${\displaystyle \begin{array}{l}{\displaystyle \mathrm{\%}\mathrm{P}\mathrm{h}\mathrm{a}\mathrm{g}\mathrm{o}\mathrm{c}\mathrm{y}\mathrm{t}\mathrm{o}\mathrm{s}\mathrm{i}\mathrm{s}\mathrm{e}\mathrm{f}\mathrm{f}\mathrm{e}\mathrm{c}\mathrm{t}=\frac{\mathrm{N}\mathrm{e}\mathrm{t}\mathrm{b}\mathrm{a}\mathrm{s}\mathrm{e}\mathrm{l}\mathrm{i}\mathrm{n}\mathrm{e}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{d}\mathrm{i}\mathrm{n}\mathrm{g}}{\mathrm{N}\mathrm{e}\mathrm{t}\mathrm{d}\mathrm{a}\mathrm{y}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{d}\mathrm{i}\mathrm{n}\mathrm{g}}\times 100\mathrm{\%}}\end{array}}$$

Vaccine-specific serum immunoglobulin

Vaccine-specific serum immunoglobulin G (IgG) concentrations following vaccination for Rabies were analyzed by indirect ELISA (Rabies Virus Antibody IgG ELISA Kit, Aviva Systems Biology, San Diego, CA). The microtiter plate provided in the kit was pre-coated with rabies virus antigen. Serum samples in serial dilution were added to the wells with anti-dog IgG conjugated Horseradish Peroxidase (HRP) and incubated for 30 min. Bound immunoglobulins were detected using tetra-methylbenzidine (TMB), and absorbance was measured using a Bio-Tek microplate reader at 450 nm. Optical density values were expressed as area under the curve (AUC) and as AUC fold-change from their respective baseline values. The AUC was calculated using commercially available software (GraphPad Prism 5 Software, GraphPad Software, Inc., San Diego, CA), using the trapezoid rule, allowing the analysis of different regions as a fraction of the total area.

Fecal IgA and calprotectin

Separate aliquots of fresh fecal samples (500 mg) were extracted using 1.5 mL of an extraction buffer and 25 µL Phenylmethanesulphonyl fluoride (Millipore Sigma, St Louis, MO). Following centrifugation at 1,500 × g for 10 min at 4 °C, supernatants were collected and analyzed for total IgA using a commercial ELISA kit (Immunology Consultants Laboratory, Inc., Portland, OR), and calprotectin using a commercial ELISA kit (MyBioSource, Inc., San Diego, CA).

ATTD of macronutrients

The diet was subsampled and ground to 2 mm particle size using a Wiley Mill (model 4; Thomas Scientific, Swedesboro, NJ). All fecal samples from the collection phase were composited for each dog and dried at 55 °C in a forced-air oven before being ground through a 2 mm screen using the same Wiley Mill that was used to grind the experimental diets. Ground samples of the experimental diets and composited dried feces were evaluated for DM and ash according to AOAC (2006; methods 934.01 and 942.05) with organic matter calculated by difference. The methods by the American Association of Cereal Chemists (1983) and Budde (1952) were used to evaluate the acid-hydrolyzed fatacid-hydrolyzed fat (AHF) content in both the diet and fecal samples. A measure of total nitrogen was completed following AOAC (2006; method 992.15) via LECO (TruMac N, Leco Corporation, St. Joseph, MI) and used to calculate crude protein content according to the Official Method of AOAC International (2006). Parr 6200 calorimeter (Parr Instruments Co., Moline, IL) was used to evaluate the gross energy of the diets and feces. Analysis of fecal and diet TDF content, as well as soluble dietary fiber and IDF content of the diets, were accomplished according to Prosky et al. (1992) and the Official Method by AOAC International (2006; Methods 985.29 and 991.43).

SCFA, BCFA, phenols, indoles, and ammonia

For analysis of SCFA and SCFA, 5 g of feces were collected in a 30 mL Nalgene bottle containing 5 mL of 2 N hydrochloric acid and stored at −20 °C. Fecal SCFA and BCFA concentrations were determined using aliquoted fresh fecal samples by gas chromatography according to a modified method by Sunvold et al. (1995). A Hewlett-Packard (HewlettPackard, Avondale, PA) Model 5890A gas chromatograph equipped with a flame ionization detector on a column (1.8 m × 4 mm i.d.) packed with GP 10% SP-1200/1% H3PO4 on 80/100 chromosorb W AW (Supelco, Bellefonte, PA) was used to evaluate the diluted fecal samples for SCFA concentration using nitrogen at a flow rate of 45 mL/min as the carrier gas. The temperatures were 125, 175, and 180 °C for the oven, injection port, and detector port, respectively. Gas chromatography was also used to determine the fecal phenol and indole concentrations following the modified method of Flickinger et al. (2003). A Thermo Scientific TRACE 1,300 Gas Chromatograph coupled with a flame ionization detector was used for this analysis, and a 1 µL sample was injected at 220 °C at splitless mode. The phenolic compounds were separated using a Nukol Supelcol column (60 m length, 0.32 mm diameter) with a film thickness of 0.25 µm. For 1 min, the oven temperature was held at 150 °C and then increased at 25 °C/min to 200 °C. The temperature was then held constant for 35 min. 5-methylinodle was used as the internal standard, and samples were analyzed in duplicate. Fecal ammonia concentration was evaluated using Chaney and Marbach’s method (1962).

Fecal DNA extraction, amplification, sequencing, and bioinformatics

A Mo-Bio PowerSoil kit (MO BIO Laboratories, Inc., Carlsbad, CA) was used to extract the total DNA from the fresh fecal samples, and the extracted DNA concentration was quantified using a Qubit 3.0 Fluorometer (Life Technologies, Grand Island, NY). A Roche High Fidelity Fast Start Kit (Roche, Indianapolis, IN) and a Fluidigm Access Array (Fluidigm Corporation, South San Francisco, CA) were used to amplify the 16S rRNA gene. Following the PacBio protocol, full-length 16S PacBio (Pacific Biology, Menlo Park, CA) primers, forward (AGRGTTYGATYMTGGCTCAG) and reverse (RGYTACCTTGTTACGACTT), were added. A Fragment Analyzer (Advanced Analytics, Ames, IA) was used to evaluate the quality of the amplicons’ regions and sizes, and equimolar amounts of amplicons from each sample were used to create a DNA pool. A 2% agarose E-gel (Life Technologies) was used to select pooled samples according to size. A Qiagen gel purification kit (Qiagen, Valencia, CA) was then used to extract the samples. The remaining products were evaluated using an Agilent Bioanalyzer to determine the profile and mean size. PacBio sequencing was completed by The Roy J. Carver Biotechnology Center at the University of Illinois. The 2× Roche KAPA HiFi Hot Start Ready Mix (Roche, Willmington, MA) and full-length 16S primers with barcodes from PacBio were used to create the 16S amplicons that were then pooled and entered into a library using the SMRTBell Express Template Prep kit 2.0 (Pacific Biology). Sequencing was performed on 1 SMRT cell 8M in Sequel II using a 10 h movie time and the circular–consensus–sequencing mode. SMRT Link V8.0 was used to evaluate circular–consensus–sequencing with the following parameters: minimum length 1,200, maximum length 2,000, minimum passes 3, and minimum rq 0.99.

The obtained sequences were analyzed using DADA2 (version 1.14; Callahan et al., 2016), and 2,091 taxa were entered into the phyloseq R page (McMurdie and Holmes, 2013). Mitochondrial DNA and taxa with no assigned phylum or zero counts were removed from the analysis in addition to the phyla Campilobacterota, Deferribacterota, and Spirochaetota due to their low prevalence in less than two samples were discarded. ASVs were converted from abundances to proportions and evaluated for the Bray–Curtis dissimilarity (Bray and Curtis, 1957). Observed ASV, Chao1, Shannon, Faith’s PD, Simpson, and Inverse Simpson indexes were used to evaluate α-diversity. The R package, DESeq2 (Love et al., 2014), was used to evaluate the differential abundance of taxa between treatments. Statistical significance was stated at a false discovery rate (Benjamini and Hochberg, 1995) <0.05. The vegan R package was used to perform Canonical Correspondence Analysis (Oksanen et al., 2019) with fecal acetate, propionate, butyrate, phenols, and indoles as the constraining variables.

Skin and coat quality assessment

Skin and coat were scored by blinded researchers according to Rees et al. (2001) using the scales below on days 0 and 28 of the experimental period.

Hair condition score: (1) dull, coarse, dry; (2) poorly reflective, non-soft; (3). medium reflective, medium soft; (4) highly reflective, very soft; (5) greasy.

Skin condition score: (1) dry, (2) slightly dry, (3) normal, (4) slightly greasy, (5). greasy.

Coats were assessed for the attributes of gloss, softness, optimum coat feel, and scale (or dander) according to Marsh et al. (2000):

• 1 Gloss (reflected light) was evaluated before touching the animal so that the texture of the coat did not influence the panelist.

• 2 Softness was assessed by the panelists running their fingers through the full thickness of the whole coat.

• 3 Optimum coat fell was assessed at the same time as softness. An optimum coat feel was considered to be an absence of a greasy or dry feeling to the coat.

• 4 Scale or dander was assessed in the three areas, which have been visually sectioned by the panelist. Assessments were carried out section by section, lifting the hairs in the opposite direction of growth and examining the skin base of the hair shafts for signs of flaking.

All of the above attributes were scored on a nine-point scale from one to five (graduated by divisions of 0.5 units), where 1 represented a coat in poor condition (e.g., excessive scale) and five a coat in good condition (e.g., no scale).

After scoring, measurements of transepidermal water loss (TEWL), hydration status, and sebum concentration were determined at the base of the inner concave face of the left and right pina, as well as on the upper back (T2-T3 between the top of scapular ridges) using standard clipping procedures (i.e., according to clipper manufacturing instructions, using a #40 blade size, shaving the designated area 48 h prior to determination of skin measurements). The body sites chosen include one area sparsely haired that does not require hair clipping prior to skin measurements being conducted and a hair-dense site that requires hair clipping for accurate skin measurements. This approach provided a more robust read-out of the potential benefits of the dietary treatments on skin and coat parameters in contrast to using only one body site. These sites have also been shown in previous literature to provide reliable results. The outcomes of interest listed above were measured using the following equipment using noninvasive methods: (1) Transepidermal water loss (TEWL): Tewameter TM 300 MDD (Courage + Khazaka Electronic GmbH, Cologne, Germany), (2) Hydration status: probe Corneometer CM 825 (Courage + Khazaka Electronic GmbH, Cologne, Germany), and (3) Sebum concentrations: external Sebumeter SM 815 (Courage + Khazaka Electronic GmbH, Cologne, Germany). A total of three readings per body site listed were taken per dog on days 0 and 28 of each experimental period.

Statistical analysis

All data were analyzed using SAS (SAS Institute Inc., version 9.4, Cary, NC) through PROC MIXED with the dog as the random effect and with d as a repeated measure to determine the effect of treatment by day. The normality of residuals was analyzed using PROC UNIVARIATE. Differences among treatments were determined using a Fisher-protected least significant difference test with a Tukey adjustment to control for type-1 experiment-wise error.

All treatment least squares means were compared using preplanned contrasts that tested for linear, quadratic, and cubic effects of GL supplementation and comparing no GL supplementation (control) to all levels of supplementation with the data from day 0 analyzed as the response variable. A probability of P ≤ 0.05 was accepted as statistically significant, and the reported pooled SEM was determined according to the Mixed Models procedure of SAS. Additionally, a probability of 0.05 < P < 0.10 was designated as a trend.

Results

Contrasts

Contrasts such as linear, quadratic, and cubic effects of GL supplementation and comparing no GL supplementation (control) to all levels of supplementation were analyzed for all the parameters included in the materials and methods section. Only the ones with statistical significance were included in the results section.

Serum chemistry

Serum chemistry was analyzed throughout the study to monitor the health status of the dogs, ensuring that the treatment supplementation would not cause any detrimental health effects. Serum metabolites were within reference ranges for all dogs except for alkaline phosphatase total, which was slightly higher than normal. However, treatment did not affect any analyzed serum metabolites or blood cell counts. There was, however, a trend (P = 0.0634) for creatine phosphokinase (CPK) being lower in dogs fed 15 mg/kg of GL when compared with dogs fed 10 mg/kg of GL (Table 1). Serum chemistry results were similar among groups at day 0.

Table 1.

Serum chemistry of adult canines fed treatments containing Ganoderma lucidum

		Treatment													Statistics
		Control			5 mg/kg			10 mg/kg			15 mg/kg			SEM1	Type 3 fixed effects
Item	Reference range2	Day 0	Day 14	Day 28	Day 0	Day 14	Day 28	Day 0	Day 14	Day 28	Day 0	Day 14	Day 28	T × D	Trt	Day	T × D
Creatinine, mg/dL	0.5 to 1.5	0.54	0.56	0.55	0.60	0.58	0.57	0.62	0.62	0.61	0.55	0.60	0.60	0.0375	0.6211	0.5263	0.2187
BUN, mg/dL3	6.0 to 30.0	15.40	15.40	15.80	16.30	16.10	14.30	16.00	15.00	16.80	15.30	16.10	14.50	1.3076	0.9817	0.7996	0.3716
Total protein, g/dL	5.1 to 7.0	6.04	6.04	6.05	6.28	6.20	6.24	5.96	6.03	6.01	6.28	6.26	6.30	0.2045	0.3970	0.9002	0.8881
Albumin, g/dL	2.5 to 3.8	3.19	3.16	3.23	3.25	3.11	3.22	3.04	3.02	3.10	3.16	3.11	3.25	0.1116	0.4829	0.0001	0.3400
Globulin, g/dL	2.7 to 4.4	2.85	2.88	2.82	3.03	3.09	3.02	2.78	2.89	2.78	3.12	3.15	3.05	0.1430	0.3762	0.0169	0.9435
Albumin/Globulin ratio	0.6 to 1.1	1.14	1.10	1.16	1.08	1.03	1.10	1.04	0.98	1.07	1.04	1.02	1.10	0.0609	0.6399	0.0001	0.9067
Ca, mg/dL	7.6 to 11.4	9.86	9.89	10.03	10.21	10.02	10.16	9.71	9.73	9.80	10.12	10.20	10.36	0.1882	0.2429	0.0413	0.5026
P, mg/dL	2.7 to 5.2	4.26	4.86	4.56	4.55	4.37	4.56	4.43	4.57	4.61	4.67	4.83	4.73	0.2881	0.9041	0.2815	0.4388
Na, mmol/L	141 to 152	144.90	145.40	144.80	145.30	145.50	145.30	144.40	145.50	145.20	145.28	146.20	145.72	0.6294	0.6450	0.0117	0.7000
K, mmol/L	3.9 to 5.5	4.41	4.50	4.59	4.42	4.49	4.62	4.51	4.50	4.76	4.43	4.51	4.49	0.1125	0.8737	0.0002	0.3643
Na:K ratio	28 to 36	33.00	32.40	31.60	33.20	32.40	31.60	32.60	32.60	30.80	33.00	32.40	32.90	0.8449	0.9156	0.0011	0.2582
CLO, mmol/L	107 to 118	110.90	111.00	110.50	108.90	109.60	109.60	109.60	110.20	109.90	109.60	109.00	109.00	0.8609	0.4951	0.7090	0.5416
Glucose, mg/dL	68 to 126	91.20	94.80	95.20	94.30	97.00	94.80	98.70	99.80	96.30	89.30	90.80	93.60	3.3376	0.3514	0.3624	0.7085
Apt, U/L	7 to 92	104.20	113.20	108.00	77.20	73.20	76.90	59.10	55.60	56.80	71.00	60.70	63.80	29.6222	0.3363	0.1705	0.4758
ALT, SGPT, U/L	8 to 65	60.90	58.06	53.10	74.30	54.30	69.90	46.33	47.00	41.33	68.50	60.60	69.40	10.0346	0.3216	0.2028	0.4398
GGT, U/L	0 to 7	3.70	3.90	3.70	4.60	4.00	4.20	3.51	3.90	4.18	3.70	3.60	3.80	0.5995	0.8056	0.8132	0.4779
Total bilirubin, mg/dL	0.1 to 0.3	0.10	0.10	0.11	0.10	0.10	0.10	0.11	0.11	0.12	0.11	0.10	0.11	0.0087	0.5555	0.2682	0.9250
CPK, U/L 4	26 to 310	85.30	74.70	89.70	100.40	85.90	93.10	111.50	83.00	97.40	86.30	74.20	77.00	6.4136	0.0483	0.0001	0.3729
Cholesterol total, mg/dL	129 to 297	191.10	198.60	204.90	244.90	241.50	241.90	215.90	205.40	213.80	223.10	230.80	242.20	38.5000	0.5318	0.0629	0.3833
Triglycerides, mg/dL	32 to 154	88.00	69.40	72.20	138.80	114.30	89.70	103.60	74.40	74.40	117.10	117.00	95.40	17.3551	0.2342	0.0002	0.4765
Bicarbonate, mmol/L	16 to 24	19.20	20.10	17.60	19.30	19.80	17.40	20.50	20.90	19.30	21.10	21.40	19.70	0.7083	0.1184	0.0001	0.7958
Anion gap	8 to 25	19.10	19.00	21.40	21.50	20.50	22.90	18.80	19.00	20.80	19.60	20.30	22.50	0.7790	0.1424	0.0001	0.5577

Table 1 (cont.). Serum chemistry of adult canines fed treatments containing Ganoderma lucidum.

¹Pooled SEM.

²University of Illinois Veterinary Diagnostic Laboratory reference ranges.

³BUN, blood urea nitrogen; CLO, Chlorine; APT, alkaline phosphatase; ALT, alanine transaminase; GGT, gamma-glutamyl transferase; CPK, creatine phosphokinase.

⁴Group T10 mg/kg tended to differ from T 15 mg/kg (P < 0.07).

Immune cell populations

Dietary treatment groups did not affect (P > 0.05) NK cells and AP cell populations of PBMC. There was a trend (P = 0.0502) that the percentage of MHC-II from B cells was greater in dogs fed 15 mg/kg of GL (41.91%) in contrast with the control group (34.63%; Table 2). A quadratic treatment effect across days (P = 0.0217) was observed for the percentage of MHC-II from B cells (Table 2). However, this treatment effect was no longer present (P > 0.05) when a change from the baseline of the percentage of MHC-II from B cells was evaluated (Table 3). No significant differences (P > 0.05) were noted among treatment groups in the values of stimulated and control PBMC T-cell populations (Table 4).

Table 2.

Natural killer and antigen-presenting cell populations of peripheral blood mononuclear cells (PBMC) in adult dogs fed diets containing G. lucidum

	Treatment													Statistics
	Control			5 mg/kg			10 mg/kg			15 mg/kg			SEM1	Type 3 fixed effects
Item	Day 0	Day 14	Day 28	Day 0	Day 14	Day 28	Day 0	Day 14	Day 28	Day 0	Day 14	Day 28	T × D	Trt	Day	T × D
Natural killer cells, %	18.12	18.25	18.42	13.35	10.75	15.80	16.16	14.95	18.42	17.33	15.04	16.62	1.6979	0.2747	0.0032	0.2621
Antigen-presenting cells
B cells, %	9.04	10.17	10.40	9.20	9.55	9.30	9.27	10.39	9.91	11.00	12.82	12.78	1.3767	0.3597	0.1077	0.9523
Monocytes, %	21.34	15.26	14.61	21.47	22.61	14.33	28.53	20.86	19.03	17.49	12.64	13.71	3.2819	0.1955	0.0002	0.3066
MHC-II from B cell, %2*	41.92	30.51	31.48	50.47	31.52	34.70	40.81	44.50	34.98	49.12	40.27	36.35	3.5876	0.0658	0.0001	0.1424
MHC-II from Monocytes, %	39.98	54.20	40.50	49.05	53.51	50.35	49.65	45.71	56.04	52.52	48.48	43.49	5.4288	0.7040	0.5962	0.1091

¹Pooled SEM.

²There is a trend that T control differs from T 15 mg/kg (P < 0.07).

^*There is a quadratic treatment effect across days (P = 0.0217).

Table 3.

Change from baseline of antigen-presenting cell populations of peripheral blood mononuclear cells (PBMC) in adult dogs fed diets containing G. lucidum

	Treatment									Statistics
	Control		5 mg/kg		10 mg/kg		15 mg/kg		SEM1	Type 3 fixed effects
Item	Day 14	Day 28	Day 14	Day 28	Day 14	Day 28	Day 14	Day 28	T × D	Trt	Day	T × D
Natural killer cells, %	0.13	0.29	−2.60	2.45	−1.21	2.26	−2.30	−0.71	1.4232	0.644	0.0020	0.1443
Antigen-presenting cells
B cells, %	1.13	1.36	0.35	0.10	1.12	0.64	1.81	2.19	1.1838	0.7045	0.9545	0.9188
Monocytes, %	−6.07	−6.73	1.13	−7.15	−7.67	−9.50	−4.85	−3.78	3.2887	0.5634	0.1082	0.1435
MHC-II from B cell, %	−11.42	−10.44	−18.95	−15.77	3.68	−5.83	−8.86	−12.78	5.6121	0.1837	0.3059	0.2079
MHC-II from monocytes, %	14.21	0.52	4.46	1.30	−3.94	6.39	−4.04	−9.03	6.0475	0.2785	0.4001	0.1097

¹Pooled standard error of the mean.

Table 4.

PBMC T-cell populations of dogs supplemented G. lucidum

	Treatment													Statistics
	Control			5 mg/kg			10 mg/kg			15 mg/kg			SEM1	Type 3 fixed effects
Item	Day 0	Day 14	Day 28	Day 0	Day 14	Day 28	Day 0	Day 14	Day 28	Day 0	Day 14	Day 28	T × D	Trt	Day	T × D
Control
T cell, % of lymphocyte	48.7	56.7	53.7	48.2	50.6	47.2	46.9	53.4	56.8	49.8	49.6	47.7	3.6853	0.6530	0.0619	0.2083
Helper T cell, % of lymphocyte	37.2	36.9	37.9	44.9	45.3	42.4	44.4	42.8	41.6	39.2	39.5	38.2	2.5327	0.1200	0.3580	0.8041
Cytotoxic T cell, % of lymphocyte	41.1	41.9	38.3	33.2	33.9	31.1	31.7	34.8	33.9	34.9	34.7	34.9	3.0015	0.1808	0.3268	0.7510
IFN-ϒ secreting helper cell, % of lymphocyte	1.68	1.64	1.60	1.80	1.76	1.54	1.23	1.75	1.55	2.30	2.10	2.57	0.57	0.8763	0.1772	0.2570
IFN-ϒ secreting cytotoxic cell, % of lymphocyte	0.12	0.07	0.17	0.23	0.32	0.18	0.05	0.12	0.05	0.06	0.044	0.12	0.0895	0.8685	0.9325	0.2035
Stimulation2
T cell, % of lymphocyte	84.04	78.13	79.43	84.24	76.72	80.25	82.79	76.36	79.88	78.81	73.64	76.55	2.0774	0.3256	< 0.0001	0.9183
Helper T cell, % of lymphocyte	31.20	30.78	27.39	33.34	34.57	27.39	32.56	32.15	31.35	32.20	31.87	27.85	2.4914	0.8678	0.0011	0.6323
Cytotoxic T cell, % of lymphocyte	26.55	25.32	23.20	22.65	21.41	19.81	20.88	22.12	20.59	21.85	21.98	22.09	1.8498	0.3524	0.0362	0.3335
IFN-ϒ secreting helper cell, % of lymphocyte	48.07	56.21	49.35	48.66	50.32	44.87	45.35	54.24	46.64	53.79	59.50	48.40	5.5750	0.8285	0.0061	0.9005
IFN-ϒ secreting cytotoxic cell, % of lymphocyte	32.41	37.96	30.87	31.87	35.55	29.16	28.28	32.56	29.44	33.83	35.29	32.03	4.1328	0.8705	0.0236	0.9599

¹Pooled standard error of the mean.

²Cells were stimulated with a cell stimulation cocktail (phorbol 12-myristate, 13-acetate, ionomycin, brefeldin A, and monoensin) for 4 h.

Responsiveness of leukocytes to TLR agonists

No significant differences (P > 0.05) were noted among treatment groups in the values of TNF-α concentrations in response to cell culture toll-like receptor stimulations. There was, however, a trend (P = 0.0724) that the responsiveness to the TLR7/8 against R848 was higher in the group control on day 28 when compared to groups control days 0 and 14, 5 mg/kg of GL days 0, 10 mg/kg days 0, and 15 mg/kg day 0 (Table 5). Responsiveness of leukocytes to TLR agonists was similar among groups at day 0.

Table 5.

TNF-α concentrations (pg/mL) in cell culture supernatant of dogs supplemented with Ganoderma lucidum

	Treatment													Statistics
	Control			5 mg/kg			10 mg/kg			15 mg/kg			SEM1	Type 3 fixed effects
Item	Day 0	Day 14	Day 28	Day 0	Day 14	Day 28	Day 0	Day 14	Day 28	Day 0	Day 14	Day 28	T × D	Trt	Day	T × D
Control	160	155	164	160	164	163	146	165	158	149	158	160	5.2201	0.2858	0.1012	0.4767
Poly I:C2 (TL3 agonist)	1,240	2,119	2,002	946	1,618	1,370	1,269	2,487	1,751	998	1,504	902.4	418.4200	0.3109	0.0047	0.8492
Lipopolysaccharide (TLR4 agonist)	773	1,138	1,414	715	850	917	901	1,669	1,644	1,037	869	757	300.3713	0.3093	0.1144	0.3213
R848 (resiquimod; TLR7/8 agonist)	11,403b	13,443b	21,835a	12,329b	15,476ab	13,742ab	12,347b	15,767ab	15,889ab	1,2937b	16,705ab	17,636ab	1,855.5502	0.6914	0.0003	0.0724
Zymozan (TLR2 agonist)	3,272	41,143	4,345	2,630	4,256	2,462	2,829	4,822	3,903	2,779	5,800	3,746	745.8301	0.5179	0.0009	0.5812

¹Pooled standard error of the mean.

²Poly (I:C); polyinosinic:polycytidylic acid.

^(a,b)Means in the same row with different superscripts tend to differ (P = 0.0724).

Phagocytosis capacity

There was a trend (P = 0.0753) that the phagocytosis response was higher in dogs fed 15 mg/kg of GL from days 14 to 28 (Table 6). A quadratic treatment trend (P = 0.0880) was observed for the phagocytosis response (Table 6).

Table 6.

Phagocytosis response in adult dogs fed diets containing Ganoderma lucidum

	Treatment									Statistics
	Control		5 mg/kg		10 mg/kg		15 mg/kg		SEM1	Type 3 Fixed Effects
Item	Day 14	Day 28	Day 14	Day 28	Day 14	Day 28	Day 14	Day 28	T × D	Trt	Day	T × D
Phagocytosis response %*	93.23b	122.58ab	85.65b	109.03b	89.08b	127.83ab	79.33b	161.84a	10.1590	0.1123	0.0001	0.0753

¹Pooled standard error of the mean.

^(a-b)Means in the same row with different superscripts tend to differ (P = 0.0753).

^*There is a quadratic treatment trend across day (P = 0.0880).

Vaccine-specific serum immunoglobulin

The vaccine-specific serum immunoglobulin G (IgG) concentrations, following vaccination for rabies in adult dogs, fed 15 mg/kg of GL were higher (P = 0.0384) than the group control when analyzed as a fold change from baseline (Tables 7 and 8, and Figure 1).

Table 7.

Vaccine-specific serum immunoglobulin G (IgG) optical density area under the curve (AUC)

	Treatment													Statistics
	Control			5 mg/kg			10 mg/kg			15 mg/kg			SEM1	Type 3 fixed effects
Item	Day 0	Day 14	Day 28	Day 0	Day 14	Day 28	Day 0	Day 14	Day 28	Day 0	Day 14	Day 28	T × D	Trt	Day	T × D
AUC	36.66b	89.36a	50.20b	37.02b	92.10a	65.88b	37.52b	93.90a	62.27b	33.01b	130.83a	62.08b	9.8620	0.5399	0.0001	0.0427

¹Pooled standard error of the mean.

^(a-b)Means in the same row with different superscripts are different (P = 0.0427).

Table 8.

Vaccine-specific serum immunoglobulin G (IgG) optical density area under the curve (AUC) fold change from baseline

	Treatment									Statistics
	Control		5 mg/kg		10 mg/kg		15 mg/kg		SEM1	Type 3 fixed effects
Item	Day 14	Day 28	Day 14	Day 28	Day 14	Day 28	Day 14	Day 28	T × D	Trt	Day	T × D
AUC fold change from baseline	1.50b	0.24c	1.72ab	1.07bc	1.71ab	0.80bc	3.21a	0.93bc	0.3792	0.0763	0.0001	0.0384

¹Pooled standard error of the mean.

^(a-c)Means in the same row with different superscripts are different (P = 0.0384).

Figure 1.

Vaccine-specific serum immunoglobulin G (IgG) fold change from baseline.

Fecal IgA and calprotectin

Supplementation of GL did not affect (P > 0.05) fecal IgA concentrations of dogs (Table 9). The fecal calprotectin concentration of dogs fed 10 mg/kg of GL was greater (P < 0.05) than the control group. A linear treatment trend across days (P = 0.0748) and a quadratic treatment effect across days (P = 0.0130) were observed for fecal calprotectin concentration (Table 9). However, when the change from baseline was evaluated, this difference disappeared because the treatments had different baseline values. Statistical analysis considering the difference from baseline revealed that dogs fed 5 mg/kg of GL tended to have greater (P < 0.0530) concentrations of fecal calprotectin in contrast with dogs fed 15 mg/kg of GL (Table 10).

Table 9.

Fecal IgA and calprotectin concentrations of adult canines fed treatments containing Ganoderma lucidum

	Treatment									Statistics
	Control		5 mg/kg		10 mg/kg		15 mg/kg		SEM1	Type 3 fixed effects
Item on fecal DM basis	Day 0	Day 28	Day 0	Day 28	Day 0	Day 28	Day 0	Day 28	T × D	Trt	Day	T × D
Fecal IgA mg/g	22.5	15.6	25.2	17.0	21.2	15.4	19.2	20.1	3.864	0.2877	0.0064	0.7475
Fecal Calprotectin µ/g2*	0.1b	0.2b	0.2b	0.5a	0.4a	0.5a	0.3ab	0.2b	0.071	0.0233	0.0032	0.0609

¹Pooled standard error of the mean.

²Control group differs from the 10 mg/kg group (P < 0.05).

^*There is a linear treatment trend across days (P = 0.0748) and a quadratic treatment effect across days (P = 0.0130).

Table 10.

Fecal IgA and Calprotectin difference from baseline of adult canines fed treatments containing Ganoderma lucidum

	Treatment
Item on fecal DM basis	Control	5 mg/kg	10 mg/kg	15 mg/kg	SEM1	P-Value
Fecal IgA, mg/g	−6.86	−8.28	−6.53	0.97	5.4573	0.6186
Fecal Calprotectin, µ/g2	0.10ab	0.29a	0.16ab	−0.02b	0.0807	0.0530

¹Pooled SEM.

^{2 (a-b)} Means in the same row with different superscripts tend to differ (P = 0.0530).

Apparent total tract macronutrient digestibility

The supplementation of GL did not affect the macronutrient digestibility (P > 0.05). The diet was highly digestible, with ATTD >80% for all analyzed macronutrient categories (Table 11). A linear treatment trend was observed for total dietary fiber (P = 0.0591; Table 11).

Table 11.

Apparent total tract macronutrient digestibility of adult dogs fed diets containing Ganoderma lucidum

	Treatment
Nutrient digestibility	Control	5 mg/kg	10 mg/kg	15 mg/kg	SEM1	P-Value
Dry matter, %	84.15	84.59	82.86	83.07	0.8216	0.3932
Organic matter, %	87.86	88.09	86.73	87.03	0.6479	0.3973
Crude protein, %	84.29	84.50	82.45	82.83	1.0921	0.4575
Acid hydrolyzed fat, %	92.12	92.23	91.52	91.44	0.5160	0.6033
Total dietary fiber, % *	53.50	50.53	46.13	46.39	3.9662	0.4919
Gross energy, %	87.62	87.91	86.32	86.71	0.6884	0.3095

¹Pooled standard error of the mean.

^*There is a linear treatment trend (P = 0.059).

Fecal fermentative end-products

No significant difference was observed (P > 0.05) among treatments regarding fecal pH and fecal scores (Table 12). Similarly, fecal concentrations of SCFA did not differ (P > 0.05) among treatments. However, dogs fed 15 mg/kg of GL tended to have a greater fecal concentration of indoles (P = 0.0623; 1.3 µmole/g DM basis) when compared to dogs fed 5 mg/kg of GL (0.6 µmole/g DM basis; Table 13). A quadratic treatment effect across days was observed for fecal concentration of indoles (P = 0.0164; Table 13). Fecal parameters, SCFA, and BCFA were similar among groups at day 0.

Table 12.

Fecal characteristics of adult canines supplemented with Ganoderma lucidum

	Treatment									Statistics
	Control		5 mg/kg		10 mg/kg		15 mg/kg		SEM1	Type 3 fixed effects
Item	Day 0	Day 28	Day 0	Day 28	Day 0	Day 28	Day 0	Day 28	T × D	Trt	Day	T × D
pH	5.6	6.0	5.8	5.8	5.7	5.8	5.9	6.0	2.4395	0.7101	0.1095	0.3889
Fecal score	2.9	3.2	3.2	3.0	2.8	3.0	2.9	3.1	1.7607	0.7425	0.1517	0.3769

¹Pooled standard error of the mean.

Table 13.

Fecal concentrations of fermentative-end products for adult canines fed treatments containing Ganoderma lucidum

	Treatment									Statistics
	Control		5 mg/kg		10 mg/kg		15 mg/kg		SEM1	Type 3 fixed effects
Item; µmole/g DM basis	Day 0	Day 28	Day 0	Day 28	Day 0	Day 28	Day 0	Day 28	T × D	Trt	Day	T × D
Phenols & indoles
Total phenols/indoles	4.5	5.0	4.0	4.5	3.5	4.3	5.0	5.1	0.6703	0.4106	0.2616	0.9597
Phenols	0.5	0.5	0.3	0.5	0.4	0.5	0.6	0.6	0.1142	0.4556	0.4499	0.7339
Indoles2*	1.0	1.1	0.6	0.7	0.7	0.9	1.2	1.4	0.2343	0.0623	0.3308	0.9997
SCFA3
Total SCFA	638.5	650.0	677.5	733.4	642.9	677.6	726.0	646.4	43.5074	0.5924	0.8376	0.3164
Acetate	394.8	392.0	402.7	442.8	406.6	425.3	445.2	388.1	23.9431	0.6955	0.9872	0.1396
Propionate	178.1	191.3	198.9	224.2	184.7	196.6	210.6	194.7	17.9286	0.5292	0.4488	0.6147
Butyrate	65.6	66.7	75.9	66.5	52.0	55.7	70.3	63.5	11.5759	0.6310	0.6544	0.8648
BCFA4
Total BCFA	33.6	31.9	49.3	40.2	18.9	25.06	39.21	34.3	9.3638	0.2948	0.5904	0.7674
Isobutyrate	7.4	7.8	7.8	8.1	5.7	7.03	8.60	7.8	0.9301	0.3576	0.5626	0.5720
Isovalerate	9.6	10.2	11.7	10.3	6.8	8.25	11.06	10.4	1.6945	0.3258	0.9905	0.7085
Valerate	16.6	13.9	29.8	21.8	6.4	9.79	19.56	16.1	7.2871	0.6171	0.7947	0.7172
Ammonia, mg/g DM	1.4	1.6	1.4	1.4	1.2	1.32	1.51	1.3	0.1155	0.4421	0.8605	0.2060

¹Pooled standard error of the mean.

²Trend for dogs fed 5 mg/kg of GL to differ from dogs fed 15 mg/kg of GL (P < 0.07).

³SCFA, short-chain fatty acids.

⁴BCFA, branched-chain fatty acids.

^*There is a quadratic treatment effect across days (P = 0.0164).

Fecal microbial DNA

The predominant phyla that comprised over 95% of the fecal microbial community (Figure 2) were Firmicutes, Bacteroidota, Fusobacteria, Proteobacteria, and Actinobacteria. The predominant families of the fecal microbial community were Lactobacillaceae, Erysipelotrichaceae, Peptostreptococcaceae, Lachnospiraceae, Fusobacteriaceae, and Prevotellaceae (Figure 3). At the genus level, the fecal microbial composition consisted predominantly of Ligilactobacillus, Peptoclostridium, Lactobacillus, Turicibacter, Fusobacterium, and Bacteroides (Figure 4). Differential abundance of the microbial communities, represented as a log two-fold change with a P-value lower than 0.05, indicated a decrease at the phylum Firmicutes, family Lactobacillaceae, and genus Ligilactobacillus in the group of dogs supplemented with 10 mg/kg BW compared with the treatment control (Figure 5). A similar decrease (P < 0.05) between treatments 10 mg/kg BM and control was observed for the phylum Actinobacteriota, family Bifidobacteriaceae, and genus Bifidobacterium (Figure 5). On the other hand, the differential abundance analysis revealed an increase (P < 0.05) at the phylum Firmicutes, family Lachnospiraceae, and genus Blautia in the group of dogs supplemented with 10 mg/kg BW compared with the treatment control (Figure 5). For the dogs supplemented with 5 and 15 mg/kg BW, no change in the differential abundance of any taxa was observed (P > 0.05). The α-diversity of the fecal microbial community was evaluated using observed ASVs, Chao1, Shannon, Simpson, Faith’s PD, and Inverse Simpson indexes, and no significant difference was observed among treatments (P > 0.05; Figure 6). The β-diversity of the fecal microbial community represented as Bray-Curtis distance revealed no significant differences among treatments (Figure 7). Additionally, PCoA, weighted and unweighted Unifrac distance analysis revealed no patterns according to treatment groups. Spearman correlation for metabolites and taxa (family) revealed a strong correlation between fecal BCFA and butyrate with the fecal bacterial families Eubacteriaceae, Anaerovoraceae, Veillonellaceae, Lactobacillaceae, Muribaculaceae, and Bifidobacteriaceae (Figure 8). Moreover, fecal indole appeared to be correlated with the families Eubacteriaceae, Anaerovoraceae, Rikenellaceae, and Muribaculaceae (Figure 8).

Figure 2.

Fecal microbial composition at the phylum level.

Figure 3.

Fecal microbial composition at the family level.

Figure 4.

Fecal microbial composition at the genus level.

Figure 5.

Differential abundance of microbial communities contrasting treatment 10 mg/kg and control (A. Family; B. Genus).

Figure 6.

Fecal microbial α-diversity in adult dogs fed diets containing Ganoderma lucidum on day 28.

Figure 7.

Fecal microbial β-diversity in adult dogs fed diets containing Ganoderma lucidum using PCoA, Bray-Curtis distance.

Figure 8.

Spearman correlation for metabolites and taxa (family).

Skin and coat quality assessment

Analysis of skin parameters (Table 14) indicated that hydration in the left pina area was significantly higher (P < 0.05) from days 0 to 28 for all treatments, except for the 10 mg/kg of GL group. An increase (P < 0.05) of sebum in the right pina from days 0 to 28 was observed in dogs fed 10 and 15 mg/kg of GL. There was a trend (P = 0.0805) that the transepidermal water loss in the left pina was greater in dogs fed 15 mg/kg of GL in contrast with the control group. Other skin parameters (i.e., hair condition, skin condition, glossiness, softness, greasiness, and scale) did not differ (P > 0.05) among treatments.

Table 14.

Skin parameters in adult dogs fed diets containing Ganoderma lucidum

	Treatment									Statistics
	Control		5 mg/kg		10 mg/kg		15 mg/kg		SEM1	Type 3 fixed effects
Item	Day 0	Day 28	Day 0	Day 28	Day 0	Day 28	Day 0	Day 28	T × D	Trt	Day	T × D
Hydration, right pina, AU2	23.00	32.63	22.96	29.17	19.83	28.87	18.93	28.17	2.9187	0.7048	0.0001	0.5522
Hydration, left pina, AU2	19.13b	31.53a	19.36b	29.36a	23.57ab	27.07ab	15.4b	29.58a	2.9688	0.8792	0.0001	0.0002
Sebum, left pina, µg/cm2	46.93	57.27	49.01	55.80	41.38	54.37	41.83	59.01	6.7912	0.9574	0.0001	0.3647
Sebum, right pina, µg/cm2	54.2ab	55.57ab	46.76ab	56.83ab	39.3b	65.7a	40.1b	60.2a	5.6709	0.9323	0.0001	0.0010
TEWL, left pina, g/m2/h3	7.88	7.88	8.52	8.24	9.86	8.37	11.14	11.01	0.9703	0.0805	0.1708	0.3233
TEWL, right pina, g/m2/h3	7.96	7.55	10.34	8.82	8.95	7.61	9.32	9.73	0.8323	0.2392	0.0452	0.1975
TEWL, upper back, g/m2/h3	10.08	11.63	12.05	10.50	11.78	12.83	11.71	11.56	0.8595	0.5959	0.5485	0.3111
Hair condition score4	3.13	3.20	3.55	3.45	3.46	3.33	3.43	3.01	0.1963	0.4493	0.1727	0.4535
Skin condition score5	2.87	2.97	3.08	3.10	3.00	3.10	2.90	2.73	0.1841	0.5486	0.9013	0.8115
Glossiness6	3.71	3.68	3.98	4.06	3.63	3.80	3.70	3.63	0.2181	0.5202	0.7369	0.8724
Softness7	3.52	3.65	3.66	3.84	3.62	3.63	3.60	3.46	0.2092	0.8428	0.6652	0.7473
Greasiness8	3.32	3.38	3.31	3.33	3.32	3.30	3.33	3.30	0.1933	0.9977	0.9453	0.9905
Scaling9	2.78	3.10	3.00	3.08	2.93	3.18	2.78	2.76	0.2218	0.7107	0.1430	0.6400

¹Pooled standard error of the mean.

²AU, arbitrary units.

³TEWL, transepidermal water loss.

⁴Hair condition score: (1) dull, coarse, dry; (2) poorly reflective, non-soft; (3) medium reflective, medium soft; (4) highly reflective, very soft; (5) greasy; determined according to Rees et al., 2001.

⁵Skin condition score: (1) dry, (2) slightly dry, (3) normal, (4) slightly greasy, (5) greasy; determined according to Rees et al., 2001.

⁶Glossiness (1 = very dull; 5 = very shiny); determined according to Marsh et al., 2000.

⁷Softness (1 = very brittle; 5 = very soft); determined according to Marsh et al., 2000.

⁸Greasiness (1 = very greasy; 5 = not greasy); determined according to Marsh et al., 2000.

⁹Scaling (1 = excessive scale; 5 = no scale); determined according to Marsh et al., 2000.

Discussion

GL is a medicinal mushroom used in China and Japan for hundreds of years for immunomodulating, anti-inflammation, and antitumor effects (Wu et al., 2016). According to phytochemical reports, 431 secondary metabolites were isolated from different species of GL over the last 40 yr (Baby et al., 2015); however, polysaccharides and terpenoids are the main biochemicals of interest (Ferreira et al., 2015). The β-glucans 1,3/1,6 are the predominant polysaccharides found in GL recognized as immunomodulators, and the ganoderic acids are the terpenoids attributed to antitumoral properties (Wu et al., 2016; Liang et al., 2019). There has been significant progress in GL research in recent decades; human clinical studies indicate that the bioactive compounds could be recognized as adjuvants for treating leukemia, carcinomas, hepatitis, and diabetes (Chen et al., 2017; Liang et al., 2019). However, no information is available about the effects of GL on companion animal nutrition and health. To the best of the author’s knowledge, this study represents the first to evaluate the effects of dietary supplementation of GL on PBMC, phagocytic activity, vaccine response, fecal fermentative end products, and macronutrient ATTD.

All dogs remained healthy throughout the study. The serum alkaline phosphatase total was slightly higher than the reference range. However, no clinical signs were noted with any animal throughout the experiment. As stated previously, creatine phosphokinase (CPK) values differed between 15 and 10 mg/kg treatment groups; however, differences were small, and values were well within the reference range reported for healthy adult dogs.

Immune cell populations

The immune response can be modulated by nutrients like β-glucans, which are glucose polymers that are major structural components of the cell wall of fungi (Volman et al., 2008). Epithelial barriers like those in the gastrointestinal tract are the line of defense of the innate immune system, which relies on recognizing microbial structures by pattern-recognition receptors (Volman et al., 2008). Macrophages and dendritic cells comprise nonspecific pattern-recognition receptors for complement receptor type 3, which is known as a β-glucan receptor (Hartland et al., 1994). Upon binding β-glucan, innate immune cells secrete cytokines such as interleukins and TNF, which in turn activate NK cells and influence the responses of B and T lymphocytes (Ferreira et al., 2015). No differences in percentages were observed in the longitudinal assessment of NK cells and AP cells of PBMC among dietary treatments. In contrast with the data presented herein, data from a previous study performed with a mouse model indicated that GL β-(1,3/1,6)-glucan suppressed inflammation induced by a high-cholesterol diet. In that study, the mice were orally supplemented with 100 µL of 8 mg/mL mushroom β-glucan; assuming a 25 g mouse, the supplementation dose was about 32 mg/kg, in addition to 2% cholesterol for 20 d. The group of mice supplemented with β-glucan presented an increase in spleen lymphocyte proliferation when compared with the group control (2% cholesterol and no β-glucan supplementation; Wu et al., 2016). However, it is essential to note that the previously mentioned study utilized a mouse model that is known to induce inflammation under an immune challenge with high-cholesterol. At the same time, the current experiment did not perform any dietary challenge, maintaining the animals healthy throughout the experiment. In addition to the immunological challenge, that study analyzed spleen cells because of the convenience of obtaining a large number of cells. Our study analyzed blood cells as a less invasive cell extraction. It is important to mention that immune cells can drastically differ in terms of turnover rate and function according to the anatomical compartment in which they reside (Hu and Pasare, 2013). Those differences could explain observing an increase in lymphocyte proliferation in the spleen upon β-glucan oral supplementation in the previously mentioned mouse study but not in blood circulation as in the present study.

In the present study, we observed a trend of increased MHC-II in B cells in the group of animals supplemented with 15 mg/kg BW of GL. Previous studies have suggested that MHC-II molecules may play a role in transducing signals to B cells (Mooney et al., 1990). During B-cell and T-cell interaction, MHC-II is critical for B-cell activation, proliferation, and differentiation (Katikaneni and Jin, 2019). Our data contrasts with those of Wilson et al. (2022), who reported no effects in the percentage of MHC-II in B cells of dogs supplemented with Saccharomyces cerevisiae fermentation product in the concentration of 30.1 mg/kg BW for 10 wk. It is important to note that Wilson et al. (2022) evaluated a yeast product, and the present study analyzed a fungus product.

Responsiveness of leukocytes to TLR agonists

The innate immune system can recognize pathogenic microorganisms through TLR receptors, leading to activation of innate immunity and instructing the development of antigen-specific acquired immunity (Akira and Takeda, 2004). At least 10 TLRs have been identified in dogs, each having specificity to various bacterial, fungal, and viral products (Schaefer et al., 2004; Heilmann and Allenspach, 2017). Ligation of TLRs by pathogen-associated molecular patterns agonists results in the activation of NF-κb, leading to the expression of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α; Medzhitov, 2001). In the present study, we observed an increase in TNF-α in stimulated cell cultures with TLRs 3, 7/8, and 2 agonists in response to vaccination; however, the supplementation of GL did not appear to have any additional effect on TNF-α production. In agreeance with our data, Wilson et al. (2022) did not observe any effect on TNF-α production in stimulated PBMC cell cultures with similar TLR agonists to the present study. The lack of significant differences in TNF-α concentrations among treatments could be attributed to a high biological variation in TNF-α concentrations among dogs.

Phagocytosis capacity

Macrophages are essential immune cells specialized for the phagocytosis of microbial pathogens (Wang et al., 2018). The present data suggest that GL supplementation, at the concentration of 15 mg/kg BW, tended to increase the macrophage’s phagocytic activity. Some studies demonstrated that GL fruiting bodies or GL polysaccharides could enhance phagocytosis of peritoneal macrophages in vivo and in vitro in mice models (Lin, 2005; Ahmadi and Riazipour, 2007). Our data are also in agreement with those of Paris et al. (2020), who observed an increase in canine macrophage phagocytic activity primed with β-glucans, analyzed with green fluorescent E. coli bioparticles. One difference between our experiment and the previous ones is that GL was orally supplemented. In the other studies, it was either intraperitoneally injected or utilized as a priming media for cell culture.

Vaccine-specific serum immunoglobulin

Upon primary encounter with a pathogen, immunoglobulin M (IgM) is secreted, followed by a class switching to immunoglobulin G (IgG) in the serum (Yu and Lieber, 2019). Results from the current study suggest that the dietary supplementation of GL at the concentration of 15 mg/kg increases the antigen-specific IgG response in serum compared with control 7 d after rabies vaccination. Our data agrees with those of Lin et al. (2019), who reported no increase in total serum IgG with β-glucan supplementation. It is important to mention that the previous study tested a S. cerevisiae fermentation product, and the present study tested a fungus product. The cellular responses induced by fungus or yeast β-glucans depend on their specific interactions with the surface cell receptors (Camilli et al., 2018). Additionally, Lin et al. (2019) analyzed total serum IgG concentration. In contrast, the present study reported a longitudinal evaluation of IgG with specificity for rabies antigen.

One study examined the immunostimulatory effect of β-(1,3/1,6)-D-glucan in puppies and determined an increase in vaccine-specific serum IgG compared with a control group (no β-glucan supplementation), a finding we corroborate in the current research. In that study, the dogs were orally supplemented with the veterinary syrup PleraSAN V at a daily dosage of 2 mL/5 kg of body weight for 2 mo, followed by subcutaneous vaccination against canine rabies on day 28 (Haladová et al., 2011). An increase in vaccine-specific serum IgG, in that study, infectious bursal disease vaccine, was also observed in pullet chickens supplemented with milled GL at the rate of 2 g/kg of feed for 7 d (Ogbe et al., 2008).

The heightened serum IgG values for the treatment of 15 mg/kg after 7 d of vaccination in contrast with treatment control may have been related to an observed tendency of higher concentration of major histocompatibility complex II (MHC-II) from B cells in the same treatment group. Expression of MHC-II is essential for activating B cells, which then can secrete immunoglobulins (Lin, 2005; Katikaneni and Jin, 2019).

Fecal IgA and calprotectin

Fecal IgA was not affected by GL supplementation. IgA is the most abundant immunoglobulin produced in mammals, having a crucial role in protecting mucosal surfaces against pathogenic and nonpathogenic microorganisms (Macpherson et al., 2001). A study by Lin et al. (2020) suggests that yeast β-glucans improve intestinal health after abrupt diet transition in adult dogs by increasing fecal IgA when compared with those in the control group. The lack of effect on IgA fecal concentrations with GL supplementation could be related to not having a challenge, such as an abrupt diet transition, since dietary macronutrient content is one of the critical factors for shaping overall gut health (Wernimont et al., 2020).

Calprotectin, which is an abundant cytosolic protein in neutrophils, is a vital inflammatory marker in the feces (Stříž and Trebichavský, 2004). Our data suggest that the supplementation of GL at 15 mg/kg BW concentration tended to decrease fecal calprotectin concentration. In agreement with our results, Førland et al. (2011) reported lower fecal calprotectin in human patients with ulcerative colitis after dietary supplementation of a mushroom extract rich in β-glucans compared with patients in the placebo group.

Apparent total tract digestibility

GL extract was analyzed as a potential functional ingredient in canine diets. Therefore, digestibility was performed to ensure that its inclusion in the diet would not affect the digestibility of the macronutrients. Our data suggest no observable effects of GL supplementation on ATTD. A similar study by Lin et al. (2019) did not report any measurable effects on macronutrient digestibility with increasing inclusion levels of β-glucans from S. cerevisiae fermentation product. Middelbos et al. (2007) analyzed the impact of increasing levels of spray-dried yeast cell wall β-glucan from 0.07 to 0.91 g daily dosed with gelatin capsules prior to feeding a complete and balanced extruded diet and observed no significant differences between supplemented diets and the control for any of the same macronutrients analyzed in the present study. The unchanged ATTD could be explained by the highly digestible diet, which consisted of more than 80% in the present study, comparable to the previously mentioned studies (Middelbos et al., 2007; Lin et al., 2019).

Fecal fermentative end-products and fecal score

Dietary supplementation of GL did not affect fecal pH, output, and score. These findings are in agreement with previous literature on canines that supplemented β-glucan derivatives of dietary yeast cell walls (Stercova et al., 2016; de Souza Theodoro et al., 2019). Another study, however, observed increased fecal scores in dogs supplemented with β-glucan from S. cerevisiae fermentation product (Lin et al. 2019).

The GL extract tested in this study contained ~70% β-glucans, which may have been available to intestinal microbes for fermentation. Regardless, supplementation had no observable effect on any analyzed fermentative end-products in the present study. These results agree with other canine studies that have reported unchanged fermentative end-products (Stercova et al., 2016; de Souza Theodoro et al., 2019). It is important to note that volatile fatty acids are rapidly absorbed along the intestinal tract, often resulting in difficulty in detecting alterations in fecal concentrations (Von Engelhardt et al., 1989)

However, the only exception observed was a trend for increased production of indoles in the group of dogs supplemented with 15 mg/kg of GL compared with the treatment of 5 mg/kg. Indole is the primary metabolite produced by gut bacteria from tryptophan. While it interacts with the gut epithelium, indole is a significant intracellular signal within the gut microbial system, contributing to the control of biofilm formation, cell motility, and gene expression in pathogenic bacteria such as E. coli (Lee and Lee, 2010). Some studies performed with indole oral supplementation in mice models have shown potential benefits to intestinal morphology and gut health by increasing the expression of tight junctions. (Bansal et al., 2010; Shimada et al., 2013; Zelante et al., 2013).

Fecal microbial DNA

Including dietary β-glucans in canine diets has been reported as an effective modulator of the gastrointestinal microbiota (Strompfová et al., 2021; Whittemore et al., 2021; Santos et al., 2022). As the gut microbiome has become increasingly associated with host health, importance has been placed on better understanding how β-glucans and their inclusion levels may impact the gastrointestinal microbial communities and their metabolic processes (Jayachandran et al., 2018). Previous studies assessing the effects of dietary β-glucans on canine gastrointestinal microbiota have mainly focused on β-glucans from yeast sources (Lin et al., 2019; Fries-craft et al., 2023). Currently, there is no literature that describes the effects of feeding β-glucans fungus derived, specifically from GL, on canine gastrointestinal microbial communities.

Firmicutes, Bacteroidetes, Proteobacteria, Fusobacteria, and Actinobacteria, have been reported as the dominant phyla making up the microbiota in healthy dogs (Wernimont et al., 2020). Dogs fed a 10 mg/kg diet had a significant decrease in the family Lactobacillaceae within the phylum Firmicutes and the family Bifidobacteriaceae within the phylum Actinobacteriota. Lactobacillaceae are a diverse family of lactic acid bacteria found in the gut microbiota of humans and many animals such as dogs, pigs, and rats (Paßlack et al., 2021; Huynh and Zastrow, 2023). These bacteria exhibited beneficial effects, including protection against pathogens and immune system stimulation (Huynh and Zastrow, 2023). Bifidobacteriaceae family contributes to intestinal functionality with the production of SCFA, which has been shown to enhance intestinal defense mediated by epithelial cells, thereby protecting the host against opportunistic bacteria such as E. coli (Fukuda et al., 2011; Bottacini et al., 2014). Spearman correlation for metabolites and bacterial families indicated a correlation between fecal BCFA and the SCFA butyrate with Lactobacillaceae and Bifidobacteriaceae. Although the group of dogs fed 10 mg/kg BW experienced a decrease in those bacterial families, no differences were observed in those fecal metabolite concentrations.

Dogs fed 10 mg/kg BW had significantly increased abundance of the family Lachnospiraceae compared with the treatment control. This family impacts their hosts by producing SCFA, converting primary to secondary bile acids, facilitating colonization resistance against pathogens, and promoting the immune system (Sorbara et al., 2020). The Spearman correlation for metabolites and bacterial families indicated a negative correlation between fecal BCFA and the SCFA butyrate with the family Lachnospiraceae; however, no differences were observed in the fecal concentration of those metabolites.

Although modest changes in microbial abundance were observed among dogs supplemented with GL, they were not consistent in characterizing a linear treatment effect. Moreover, alpha diversity, which provides a summary of the microbial community in individual samples in terms of richness (number of species) and evenness (how well each species is represented; Nikolova et al., 2023), as well as β-diversity, which determines whether microbial communities are significantly different (Lozupone and Knight, 2005), were not affected by GL supplementation. Reilly et al. (2021) observed greater fecal microbial β-diversity for dogs fed a diet containing 29.88% inclusion level of a dried yeast product (a β-glucan source) for 14 d. Therefore, a higher inclusion level of GL might be needed to affect the gut microbiota.

Skin and coat quality assessment

Very little is known about the cutaneous effects of dietary fungus extracts. However, β-glucans, active constituents of GL, have been reported to promote skin wound healing and mitigate postburn infection, in addition to playing a role in photoaging and skin whitening (Tie et al., 2012; Hu et al., 2019). Tie et al. (2012) showed that the oral administration of GL polysaccharides could improve skin by suppressing the cutaneous manganese superoxide dismutase nitration and mitochondrial oxidative stress.

The present study tested several skin parameters and skin and coat visual assessments; no significant differences were observed among the treatments. However, a sebum increase was noted in the right ear in the group of dogs supplemented with 10 and 15 mg/kg from days 0 to 28. The sebum increment was attributed to the fact that the dogs would lay more on their right side. This ear sebum is vital for protecting the ear by trapping dust and other foreign particles that could damage the eardrum (Lukolo et al., 2021). The lack of effect of GL in skin parameters may be correlated to the epidermal turnover time, which was calculated to be 47 to 48 d (Iizuka, 1994), and the supplementation period consisted only of 28 d.

Conclusions

The inclusion of different concentrations of GL as top-dressing before feeding had no detrimental effects on the dog’s overall health, fecal characteristics, ATTD, skin, or coat health. However, the 15 mg/kg BW dose increased serum vaccine-specific IgG response, tended to increase macrophage phagocytic activity, and tended to increase the percentage of MHC-II from B cells. Therefore, it can be concluded that GL may exert beneficial immunostimulatory effects in healthy adult dogs when provided at a daily dose of 15 mg/kg BW. In addition, higher doses might be necessary to elicit beneficial and more robust shifts in canine gut microbiota and fecal metabolites.

Glossary

Abbreviations

ALT

alanine transaminase

AP

antigen-presenting cells

ATTD

apparent total tract digestibility

AU

arbitrary units

AUC

area under the curve

BCFA

branched-chain fatty acids

BUN

blood urea nitrogen

BW

body weight

CLO

chlorine

CPK

creatine phosphokinase

FITC

anti-CD3-fluorescein isothiocyanate

GGT

gamma-glutamyl transferase

GL

ganoderma lucidum

HRP

horseradish peroxidase

IFN-γ

interferon gamma

Ig

immunoglobulin

MHC-II

major histocompatibility complex II

NK

natural killer

PBMC

peripheral blood mononuclear cells

SCFA

short-chain fatty acids

Tc

cytotoxic T-cell

TEWL

transepidermal water loss

Th

helper T-cell

TLR

toll-like receptors

TMB

tetramethylbenzidine

TNF-α

tumor necrosis factor-alpha

Conflict of Interest Statement

Maria Spindola is employed by Alura INC. All other authors have no conflicts of interest.