Animals and housing
Sixteen adult intact beagle dogs were used (eight males and eight females), with an average body weight of 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 the Research laboratory on canine nutrition of the Federal University of Parana (Curitiba, PR, Brazil) from Maiorca Kennel (Colombo, PR, Brazil) when they were 3-4 months old.
During most of the diet adaptation period (until the 16th day) dogs had free supervised access to an outdoor area for 2h a day. Between days 17-25 the dogs were individually housed at the kennels to allow for faecal collection. All dogs received extra attention and kennel enrichment during this period. The dogs will be donated when they complete 6 years of age. The use of animals for this study was approved by the Ethics Committee on Animal Use from the Agrarian Sciences Sector, 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 supplementation, and the other part (eight dogs, four males and four females) was used as the test treatment containing 62.5 mg/kg of a diet with a mixture of Bacillus subtilis (3.66x107 cfu/kg of the diet) and Bacillus licheniformis (3.66x107 cfu/kg of the 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 chemical composition of the experimental diets is shown in (Table 7).
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 (dry matter basis, %).
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
|
Crude fibre
|
2.23
|
2.42
|
Calcium
|
1.18
|
1.23
|
Phosphorus
|
0.89
|
0.91
|
DFM Direct-fed microbials (62.5 mg/kg of a diet with a mixture of 3.66x107 cfu/kg Bacillus subtilis and 3.66x107 cfu/kg of Bacillus licheniformis)
Experimental procedures
The digestibility assay followed the total faeces collection method as recommended by the Association of American Feed Control Official [30]. The diets were provided during a twenty-day 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.), in amounts sufficient to meet the animal’s metabolisable energy (ME) requirement according to the National Research Council [31], 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 previously-identified plastic containers, covered and stored in a freezer (-14°C) to be analysed 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 from each animal. Faeces were dried in a forced ventilation oven (320-SE, Fanem, São Paulo, Brazil) at 55oC for 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), crude fibre (CF, method 994.13), ether extract in acid hydrolysis (EEAH, method 954.02), and ash (942.05) were determined in both diets and faeces according to the Association of the Official Analytical Chemists [32]. 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. Nitrogen-free extract was calculated as 100 – CP – Ash – CF – EEAH.
Faecal odour 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 and covered with plastic film with holes (same number and size). The containers were classified as: A (control diet) and B (DFM diet), so the participants would not have information about the treatment. The sensorial analysis was performed by 50 evaluators with fresh faeces (up to 30 minutes after defecation) and 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 the following scoring system: 1 = better odour than control (less fetid); 2 = same as control; 3 = worse than control (more fetid).
Faecal pH and ammonia concentrations were analysed in faeces collected up to 15 minutes after defecation. Faecal pH was measured in a digital pH meter (331, Politeste Instrumentos de Teste Ltda, São Paulo, SP, Brazil) using 3.0 g of fresh faeces diluted in 30 mL of distilled water. The ammonia concentration was determined according to the method described by Brito et al. [34].
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 mixed with 30 mL of 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) for 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 for 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 analysed by chromatography using 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 a 5.0 rate. The transfer line and ionization source temperatures were maintained at 40°C and 220°C, respectively, with the 1-L injection volume in the split mode (1:10 rate). The GC oven temperature was maintained at 220°C (5 min) with a 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), with 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 analysed according to the method described by Jourdian et al. [35]. Biogenic amines were analysed according to the method described by Urrego et al. [36] in fresh faeces, collected up to 15 minutes after defecation.
The DMf, consistency score, faecal odour, pH, ammonia, phenols and indoles were also analysed in the same samples 6 hours after defecation. For the analysis performed 6 hours after defecation, faeces were maintained at room temperature (average of 24.5ºC, 84% relative air humidity and in the shade for 6 hours.)
Calculations and Statistical analyses
Based on the laboratory results, the apparent total tract digestibility (ATTD) coefficients and the diet’s ME were calculated according to the Association of American Feed Control Official [30]:
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 odour 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 analysed by the Mann-Whitney-Wilcoxon test (P <0.05). The frequency of faecal odour scores was analysed by the chi-square test (P <0.05).