Animals and experimental design: EXP1 and EXP2
All experimental procedures were performed in accordance with the guidelines of the Local Ethical Committee for Animal Research (Ministry of Science and Higher Education, Poland) as well as in compliance with the ARRIVE guidelines. The study conformed to the 28/2020 statement of Local Ethical Committee in Poznan, Poland. The dog owners gave their informed consent in writing. The research consisted of two consecutive studies. The first, EXP1, surveyed a group of eighteen healthy laboratory adult beagle dogs (n = 18, nine females and nine males, 2 years old). In EXP1, a 3 × 3 Latin square design with 3 treatments (CON, HEP, SIL) and three periods was used. Each treatment was given to six dogs (three females and three males) in a given period, giving eighteen replicates. A commercial basic diet (Addvena, Poznań, Poland) composed of lamb (including fresh lamb meat 50%), potatoes, peas, beet pulp, animal fat, potato protein, tomato puree, dried alfalfa, flaxseed, brewer’s yeast, salmon oil, sodium phosphate dihydrate, chicory root, glucosamine, and chondroitin sulphate was the control diet (CON). The first experimental diet (HEP) was commercially available feed (CON) supplemented with commercial hepatoprotectant containing silybin (Hepaxan, Vebiot, Dębica, Poland), while the second diet (SIL) was CON supplemented with pure silybin. The diet for both groups contained silybin, pure or as a preparation, at a dose of 12.75 mg per 10 kg (8.5 mg per 5 kg) body weight. EXP1 was divided into three periods, each lasting 28 days: this consisted of a 23-day adaptation phase (days 1 to 23) and a five-day total faecal collection phase (days 24 to 28), followed by a 12-day wash-out period. The experiment lasted 108 days (so each dog had three 28-day periods with 12-day wash-out periods between them). Titanium dioxide (TiO2) was included in the diets as a digestion marker at 0.2% of diet. The analysed crude nutrient concentration in the diets and dietary fatty acid profile are presented in Table 1. Each dog was housed individually in a kennel that enabled social contact among animals, was fed twice a day, and had free access to water. During the adaptation phase, the dogs had access to an outside playground for exercise and socialisation. The maintenance energy requirement (MER) was estimated according to FEDIAF [58] and the diets met the MER of the dogs. Each animal taking part in the experiment was up to date on their vaccination and deworming schedules before beginning.
EXP2 used client-owned dogs (n = 15) referred to the University Centre for Veterinary Medicine at Poznań University of Life Sciences, in which a hepatic disorder was diagnosed. The diagnostic process did not reveal a specific etiology agent, therefore these cases were considered idiopathic. A profile of the dogs taking part in EXP2 can be found in Supplementary Table 1. The criteria we established for diagnosing a hepatic disorder were a clinical demonstration of at least one of the symptoms described as most prevalent in dogs with chronic hepatitis [48], including decreased appetite, lethargy/depression, icterus, ascites, PU/PD, vomiting, diarrhoea, or subsequently an increase in at least three out of these four liver markers: alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), and gamma glutamyl transpeptidase (GGT). The exclusion criteria were infectious or parasitic diseases, systemic, neurological or traumatic diseases or general symptoms of food intolerance or allergy in the past. Moreover, individuals with confirmed hepatocarcinoma or other liver-associated cancers were excluded from the study. The dog owners were advised to begin supplementing their pets’ diet with commercially available preparation containing silybin (Hepaxan, Vebiot, Dębica, Poland) at the dose recommended by the manufacturer (Supplementary Table 2).
Health status and Body Condition Score (BCS): EXP1 and EXP2
The average weight was 18.1 kg for males and 12.9 kg for females in EXP1, and 28.7 kg for males and 22.1 kg for females in EXP 2. In EXP1, body weight was measured on days 1 and 28 of the experimental period and feed intake was recorded daily. In EXP2, body weight was measured at the beginning (day 1) and at end (day 28) of supplementation with the hepatoprotectant. For all dogs in EXP1, the body condition score (BCS) was assessed throughout the experimental period in line with the recommendations of the World Small Animal Veterinary Association [59]. The dogs in EXP1 and EXP2 underwent weekly check-ups consisting of physical examination, including rectal temperature measurement, mucous membrane inspection, heart and lung auscultation, and stomach palpation (abdominal examination). The dogs were determined to be clinically healthy if the physical examination revealed no pathological findings.
Blood sample collection: EXP1 and EXP2
Blood samples were collected via cephalic venipuncture as follows:
1) EXP1: on the last day (day 28) of each treatment period at 6.00 AM.
2) EXP2: on the first day (H1) of the supplementation and 28 days later (H28).
In both EXP1 and EXP2, blood samples were collected in two vacutainer tubes. One of these contained K3EDTA anticoagulant and was used for hematological examination; the second tube contained serum separator gel and was used to obtain serum for biochemical and miRNA examination, fatty acid profiles, and serum interleukin, immunoglobulin, and acute phase protein analysis. Blood from the second set of tubes was left at room temperature for blood clot formation and then centrifuged at 3500 rpm for 10 min at 4 °C to obtain serum. The serum samples were transferred to Eppendorf tubes, labeled, sealed, and frozen at -80 ℃ to await analysis.
Hematology and serum biochemistry analysis: EXP1 and EXP2
CBC was performed using a Vet ABC Animal Blood Counter automatic haematological analyser (ABX, Montpellier, France) with the following parameters: red blood cell count (RBC), neutrophil count (NEUT), lymphocyte count (LYM), monocyte count (MONO), eosinophil count (EOS), basophil count (BASO), hemoglobin (HEM), hematocrit (HTC), mean corpuscular volume (MCV), mean corpuscular hemoglobin (CHC), mean corpuscular hemoglobin concentration (MCHC), white blood cells (WBC), and platelet (PLT) counts.
Biochemical analysis of ALT, ALP, AST, GGT, alpha amylase, total protein, total bilirubin, cholesterol, creatinine kinase (CK), fructosamine, glutamate dehydrogenase (GLDH), glucose (GLUC), creatinine, lactic dehydrogenase (LDH), lipase (DGGR), urea, triglycerides (TG), chloride, inorganic phosphorus, magnesium, potassium, sodium, calcium, albumin, globulin, and albumin/globulin ratio was carried out using a Dade Behring Dimension RxL analyser (Siemens Healthcare Diagnostics, Newark, DE, USA).
The reference ranges used to evaluate health status for the haematological and biochemical parameters were based on the Merck Veterinary Manual [60].
Serum interleukins, immunoglobulins, and acute phase proteins analysis: EXP1
In order to determine the concentrations of selected immunological parameters, commercially available species-specific quantitative ELISA kits were used as follows: for IgA, IgG, and IgE (Wuhan Fine Biotech, China), for IgM (Signalway Antibody, MA, USA), for IL1β, IL4, IL6, IL8, and IL10 concentrations (Wuhan Fine Biotech, China), C-reactive protein (CRP) (BlueGene, Shanghai, China), serum amyloid-A (SAA) (ABclonal, Massachusetts, USA), haptoglobin (Cusabio, TX, USA). Prior to analysis, all serum samples were diluted (depending on assay range and the expected analyte concentration). For each test, serial dilutions of standards were tested in order to obtain a calibration curve, which was then computer-adjusted. From this calibration curve, the values of the unknown protein concentration samples were calculated. All analyses were performed following the manufacturer`s instructions.
Diet and faeces sample collection: EXP1
Diet samples were collected daily during the faecal collection phase (days 24 to 28) of each period and stored at -20 ℃ for further analysis. Faeces were collected daily from day 24 to 28, including at the time of daily walks. Freshly collected faeces samples were stored at -20 ℃. The total individual daily faecal output was weighed, mixed, and stored at -20℃ to await further analysis.
Chemical analysis of diets and faeces and digestibility calculation: EXP1
The faecal samples were dried for 72 hours at 55 ℃, following AOAC International guidelines [61]. The dried faeces were milled in a laboratory mill (ZM200, Retsch, Haan, Germany) using a 1 mm sieve. The chemical composition of the feed and faeces samples was analysed following AOAC [61] method no. 934.01 for dry matter, method no. 976.05 for crude protein (using a Kjel-Foss Automatic 16210 analyser), and method no. 973.18 for crude fat (using a Soxtec System HT analyser). The apparent total tract digestibility (ATTD) of individual nutrients relative to the ratio of TiO2 was calculated as a percentage based on the following equation:
Dietary and serum fatty acid (FA) profile: EXP1
FA concentration was determined using a gas chromatograph [62] with some modifications. Briefly, 3 mL of 2 M NaOH solution was added to 1 g feed or 0.5 ml serum sample, respectively, in screw-cap Teflon-stoppered tubes (glass, 15 mL) for fat hydrolysis. The hydrolysed FA samples were incubated on a block heater at 90 °C for 40 min. Analysis of fatty acids methyl ester (FAME) was performed on a gas chromatograph (GC Bruker 456-GC, Billerica, MA, USA) equipped with a capillary column (100 m fused-silica, 0.25 mm i.d., 0.25 μm film thickness; Chrompack CP7420, Agilent HP). Fatty acids were identified based on their retention times and were expressed as g/100 g FA. The observed peaks were identified by comparison of their retention times with FAME standards (37 FAME Mix, Sigma Aldrich, PA, USA) using a Galaxie Work Station 10.1 (Varian, CA).
Urine samples and urinalysis: EXP1
Free catch urine was collected on the last day of each feeding period using a Uripet urine collection device (Rocket Medical, Watford, England). Then 3 ml of urine was stored at -20 °C and analysed for creatinine, cortisol, and pH using VetLab Station (IDEXX Poland) within two weeks of sampling.
Serum miRNA expression: EXP1 and EXP2
The investigation into the effects of the test compounds on the expression of miRNA in blood serum was performed in two experiments: In EXP1, we examined the effect of commercial hepatoprotectant and silybin supplementation on miRNAs expression in healthy dogs (EXP1). In EXP2, we investigated the effect of commercial hepatoprotectant on miRNA expression in dogs with liver disorders. The healthy dogs in EXP1 were used as the control group in EXP2.
MiRNA was isolated using QIAzol Lysis Reagent and miRNeasy Serum/Plasma Kit (Qiagen, Germany). Endogenous control was added to the samples during isolation (miRNeasy Serum/Plasma Spike-In Contro; Qiagen, Germany). Following extraction and elution of RNA, the samples were immediately frozen at -80 °C. RNA content and relative purity were determined using the UV-Vis spectrophotometric method with a NanoPhotometer NP80 (Implen, Munich, Germany). Reverse transcription was performed using a miScript II RT kit, following the manufacturer’s instructions. The master mix was prepared on cooling blocks and contained 4 µl of 5× HiSpec Buffer, 2 µl of 10× Nucleics Mix, 2 µl of Reverse Transcriptase (RT), and 2 µl of RNase-free water per reaction, giving a total volume of 20 µl. 10 µl of the master mix was added to 10 µl of the total RNA extracted from serum. The reaction was performed on a Mastercycler (Eppendorf, Germany) at 37 °C for 60 minutes, followed by 95 °C for 5 minutes to inactivate the RT. The relative expression of miRNAs (miR-192, miR-122 and miR-126) was measured by real-time PCR on QuantStudio12K Flex (Applied Biosystems, USA) using specific primers (MS00029883; Cf_miR-192_1 miScript, MS00029400; Cf_miR-122_1 miScript, MS00029428; Cf_miR-126_1 miScript; Qiagen, Germany) and miScript SYBR Green PCR kit (Qiagen, Germany). SNORD72 and RNU6-2 were used as endogenous controls (MS00033719; Hs_SNORD72_11, MS00033740; Hs_RNU6-2_11; Qiagen, Germany). Relative quantification of miRNA expression was calculated with the 2-ΔΔCt method.
Data analysis
Both data sets for EXP1 and EXP2 were analysed with IBM SPSS Statistics 24 software (SPSS Inc, Armonk, USA). For each variable in EXP1 a repeated measure analysis (one-way ANOVA) with the Tukey test as post-hoc analysis was performed. A significant value was accepted at p < 0.05. In both data sets Shapiro-Wilk test was applied to assess the data normality, whereas in data from EXP1 homogeneity of variance was evaluated through Levene's test. Variables in EXP2 were compared using the dependent Student’s t-test for normally distributed variables with a significant value accepted at p < 0.05.