Animals and housing
Sixteen adult intact beagle dogs were used (eight males and eight females), average body weight being 10.3 + 1.07 kg and four years of age. All animals underwent previous clinical and physical examinations, were vaccinated, dewormed, and individually housed in covered brickwork kennels (5 meters long x 2 meters wide), containing a bed and free access to fresh water. The environment temperature ranged from 16 °C to 28 °C with a 12-h light–dark cycle (light 6 am–6 pm). All animals were brought to Research laboratory on canine nutrition of the Federal University of Parana (Curitiba, PR, Brazil) from Maiorca Kennel (Colombo, PR, Brazil), since they have 3-4 months old.
During the most of diet adaptation period (until 16th day) dogs have free access to outdoor area under supervision during 2h per day. Among days 17-25th dogs were individually housed at the kennels to allow fecal collection. All dogs received extra attention and kennel enrichment during this period. Dogs will be donated when they complete 6 years old. The use of animals for this study was approved by the Ethics Committee on Animal Use from the Sector of Agrarian Sciences, Federal University of Paraná, Curitiba, PR, Brazil (012/2019).
Experimental diets
The same commercial diet for adult dogs was divided into two parts and used in the experimental treatments. One part (eight dogs, four males and four females) was used in the control treatment, with no DFM, and the other part (eight dogs, four males and four females) was used as the test treatment, containing 62.5 mg/kg of diet of a mixture of Bacillus subtilis (3.66x107 cfu/kg of diet) and Bacillus licheniformis (3.66x107 cfu/kg of diet) as DFM (PureGro®, DSM, Heerlen Netherlands). The diet had the following composition: poultry viscera meal, meat meal, corn, soybean meal, poultry fat, swine liver hydrolysate, sodium chloride, citric acid, antioxidants (BHT, BHA), propionic acid, vitamin A, vitamin D3, vitamin E, vitamin B1, vitamin B6, vitamin B12, vitamin K3, nicotinic acid, folic acid, biotin, calcium pantothenate, zinc sulfate, calcium iodate, sodium selenite, copper sulfate, iron sulfate, manganese sulfate and zinc oxide. The experimental chemical composition of the diets is shown in (Table 7).
The DFM was diluted in poultry viscera oil and used on top of the test diet. The same amount of oil, without DFM, was used on the control treatment, ensuring that the diets were isonutritive.
Table 7 Analysed chemical composition of the experimental diets based on dry matter (%).
Item
|
Control
|
DFM
|
Dry matter
|
91.77
|
91.51
|
Crude protein
|
21.75
|
21.12
|
Ether extract in acid hydrolysis
|
9.22
|
9.04
|
Ash
|
7.02
|
7.00
|
Calcium
|
1.18
|
1.23
|
Phosphorus
|
0.89
|
0.91
|
DFM Direct-fed microbials (62.5 mg/kg of diet of a mixture of 3.66x107 cfu/kg Bacillus subtilis and 3.66x107 cfu/kg Bacillus licheniformis)
Experimental procedures
The digestibility assay followed the total faeces collection method as recommended by Association of American Feed Control Official [28]. The diets were provided during a twenty days adaptation period, followed by five days of total faeces collection, resulting in a mixture of faeces from each animal.
The food was provided twice a day (8:30 a.m. and 4:00 p.m.), the amounts being sufficient to meet the animal’s metabolizable energy (ME) according to [29], where: ME (kcal/day) = 130 x Body weight0.75. Water was provided ad libitum. The faeces were collected and weighed at least two times per day and stored in individual plastic containers previously identified, covered and stored in a freezer (-14°C) to be analyzed later.
At the end of the collection period, the faeces of each replicate were thawed at room temperature and homogenized separately, forming a composite sample of each animal. Faeces were dried in a forced ventilation oven (320-SE, Fanem, São Paulo, Brazil) at 55oC during 48 hours or until reaching constant weight. Diets and faeces were ground to 1.0 mm in a hammer mill (Arthur H. Thomas Co., Philadelphia, PA, USA), using 1.0-mm wire mesh sieves for the bromatological testing (in duplicate and with repetitions when the variation was higher than 5%).
The amounts of dry matter at 105ºC (DM105), crude protein (CP, method 954.01), ether extract in acid hydrolysis (EEAH, method 954.02) and ash (942.05) were determined in both diets and faeces according to Association of the Official Analytical Chemists [30]. The amount of gross energy (GE) was established using a calorimetric pump (Parr Instrument Co., model 1261, Moline, IL, USA), and organic matter (OM) was calculated by the difference between 100 – Ash.
The faecal odor was evaluated and scored on the 25th day of the experimental period. Faeces from three animals per treatment were randomly collected, homogenized and the same amounts (5.0 g) were placed in plastic containers of the same size, covered with plastic film with holes (same number and size). The containers were classified as: A (control diet) and B (diet with DFM), so the participants would not have information about treatment. The sensorial analysis was performed by 50 evaluators with fresh faeces (up to 30 minutes after defecation) and also six hours after defecation, with different people at each point in time. In the evaluation, sample B with DFM was compared to A (control diet), using a scoring system: 1 = better odor than control (less fetid); 2 = same as control; 3 = worse than control (more fetid).
Faecal pH and ammonia concentration were analyzed in faeces collected up to 15 minutes after defecation. Faecal pH was measured in digital pH meter (331, Politeste Instrumentos de Teste Ltda, São Paulo, SP, Brazil), using 3.0 g of fresh faeces diluted in 30 mL distilled water. The ammonia concentration was determined according to the method described by [32].
Fresh faeces collected up to 15 minutes after defecation were used to determine SCFA and BCFA. A properly labelled plastic container with a lid was used to weigh 10 g of faeces that were mixes with 30 mL 16% formic acid. This mixture was homogenized and stored at 4°C for 3 to 5 days. Before the analysis, these solutions were centrifuged at 5000 rpm (2K15 centrifuge, Sigma, Osterodeam Hans, Germany) during 15 minutes. At the end, the supernatant was separated and centrifuged. Each sample underwent three centrifugations and at the end of the last one, part of the supernatant was transferred to a properly identified eppendorff for subsequent freezing. Later on, the samples were thawed and centrifuged again at 14000 rpm during 15 minutes (Rotanta 460 Robotic, Hettich, Tuttlingen, Germany). Faecal SCFA and BCFA were determined by gas chromatography (Shimadzu®, model GC-2014, Kyoto, Japan), using a 30 m long and 0.32 mm wide glass column (Agilent Tecnologias, HP INNO cera-19091N, Santa Clara, USA). Nitrogen was used as the carrier gas at a 3.18 mL/min flow rate. Working temperatures were 200°C at injection, 240°C in the column (at a 20°C/min rate), and 250°C in the flame ionization detector.
Phenols and indoles were analyzed by chromatography, a GCMS2010 Plus gas chromatographer (Shimadzu®), coupled to a TQ8040 mass spectrometer with an AC 5000 autosampler and a split-splitless injector. Chromatographic separations were obtained in the SH-Rtx-5MS (30 m x 0.25 mm x 0.25 μm - Shimadzu®) column with a 1.0 mL min-1 flow rate, and helium as the drag gas at 5.0 rate. The transfer line and ionization source temperatures were maintained at 40°C and 220°C, respectively, the 1 L injection volume in the split mode (1:10 rate). The GC oven temperature was maintained at 220°C (5 min), with 40°C min-1 increase to 280°C (5 min). Total analysis time was 31 minutes and the mass spectrometer operated in the full scan modes (m/z = 40 to 400) and selective ion monitoring (SIM), electron ionization at 70 eV. GCMSsolution® was the software used in the data analysis.
For the sialic acid determination, faeces were lyophilized (Alpha 1-4 LO plus, Christ, Osterodeam Hans, Germany) and analyzed according to the method described by [33]. Biogenic amines were analyzed according to the method described by [34] in fresh faeces, collected up to 15 minutes after defecation.
The DMf, consistency score, faecal odor, pH, ammonia, phenols and indoles were also analyzed in the same samples 6 hours after defecation. For the analysis performed 6 hours after defecation, faeces were maintained at room temperature (average 24.5ºC and 84% relative humidity of air), in the shade during 6 hours.
Calculations and Statistical analyses
Based on the laboratory results, the coefficients of total tract apparent digestibility (ATTD) and the diets metabolizable energy (ME) were calculated according to [28]:
ATTD% = (g of nutrient intake – g of nutrient excretion)/g of nutrient intake x 100.
ME (kcal/g) = {kcal/g GE intake – kcal/g GE faecal excretion – [(g CP intake – g CP
faecal excretion) x 1.25kcal/g]} / g of feed intake.
The experiment had a completely randomized design with two treatments, each one with eight replicates, except for faecal odor that had 50 replicates. Each dog was considered an experimental unit. The Shapiro-Wilk test was used to determine normality of the data and the homoscedasticity of variances was analysed by Bartlett’s test. When these assumptions were met, the t-Student’s test was used at a 5% significance level. The non-parametric data were analyzed by Mann-Whitney-Wilcoxon test (P <0.05). Frequency of faecal odor scores was analyzed by the chi-square test (P <0.05).