The animal experimental work was carried out at the Amazcala Campus of the College of Natural Sciences, Autonomous University of Queretaro. Ethical conditions were approved using criteria established by the Ethics Advisory Committee for Animal Experimentation, University of Zaragoza [15]. The experimental protocol was approved by the Bioethics Committee of the College of Natural Sciences, Autonomous University of Queretaro (number 93FCN2016). Sixteen New Zealand rabbits aged 32-days-old were housed as groups of 4 rabbits in American type cages (90cm x 60cm x 40cm) equipped with feeders and drinking fountains. Rabbits were allocated in a 2x2 design experiment with, 4 animals per group. At the outset of the experimental work (i.e., when rabbits were 32 days old) animals were allocated to a group based on either having ERE or being clear of it (deemed healthy), and on a diet of 32% NDF which is recommended for the intestinal health of the rabbit [16], or 36% NDF which has been suggested for treatment of ERE [17]. Although it was not possible to balance animals for weight between ERE and healthy groups, due to ERE animals being significantly lighter at the outset, animals were balanced as closely as possible for dietary regimes. The diets were formulated with the same ingredients; alfalfa meal, canola meal, wheat bran, sunflower meal, sodium chloride, calcium carbonate, vitamins and minerals (Table 1). No antibiotics or anticoccidial drugs were included in the diet. All animals were given ad libitum access to food and water.
Table 1
Proximal chemical composition of diets 1.
Proximal chemical analysis
|
Diet 1 (32%)
|
Diet 2 (36%)
|
Dry weight
|
91.2
|
91.3
|
Neutral Detergent Fibre (NDF)
|
31.7
|
35.8
|
Acid Detergent Fibre (ADF)
|
23.5
|
25.6
|
Hemi cellulose
|
8.2
|
10.2
|
Crude Protein
|
15.8
|
16.0
|
Ash
|
8.8
|
8.6
|
Lignin
|
7.8
|
7.8
|
Fat Content
|
1.7
|
1.8
|
Phosphorus
|
0.85
|
0.86
|
Calcium
|
1.42
|
1.43
|
Selenium
|
0.35
|
0.35
|
Vitamin A UI/kg
|
5167
|
5147
|
Vitamin D3 UI/kg
|
974
|
974
|
Vitamin E mg/kg
|
53.3
|
55.2
|
Vitamin B12 mg/kg
|
4.3
|
4.5
|
1 Data were obtained in the Animal Nutrition Laboratory, Autonomous University of Querétaro following the AOAC manual [18] |
Table 2
Volatile fatty acid, ammonia and pH values for cecotroph samples collected. Mean and standard deviation values are shown.
Metabolite / Parameter
|
T1
|
T2
|
T3
|
T4
|
P value - Diet
|
P value - Disease
|
P value - interaction
|
Total VFA µmol/g
|
9.75 ± 0.14
|
13.35 ± 0.16
|
14.36 ± 0.19
|
10.08 ± 0.16
|
0.034
|
0.001
|
< 0.001
|
Acetic acid µmol/g
|
3.65 ± 0.10
|
4.35 ± 0.01
|
4.95 ± 0.09
|
3.82 ± 0.10
|
0.028
|
0.001
|
< 0.001
|
Propionic acid µmol/g
|
1.71 ± 0.09
|
2.68 ± 0.03
|
2.00 ± 0.10
|
1.85 ± 0.08
|
0.007
|
< 0.001
|
< 0.001
|
Butyric acid µmol/g
|
1.24 ± 0.03
|
2.76 ± 0.02
|
1.25 ± 0.02
|
1.24 ± 0.02
|
< 0.001
|
< 0.001
|
< 0.001
|
Isobutyrate µmol/g
|
0.43 ± 0.02
|
0.41 ± 0.01
|
0.97 ± 0.02
|
0.43 ± 0.02
|
< 0.001
|
< 0.001
|
< 0.001
|
Valeric acid µmol/g
|
1.02 ± 0.01
|
1.32 ± 0.01
|
2.00 ± 0.04
|
1.03 ± 0.02
|
< 0.001
|
< 0.001
|
< 0.001
|
Isovalerate µmol/g
|
0.90 ± 0.01
|
0.82 ± 0.01
|
1.27 ± 0.02
|
0.97 ± 0.01
|
< 0.001
|
< 0.001
|
< 0.001
|
Ammonia %
|
0.06 ± 0.01
|
0.04 ± 0.01
|
0.16 ± 0.07
|
0.07 ± 0.01
|
NS
|
NS
|
NS
|
pH
|
6.58 ± 0.17
|
6.46 ± 0.07
|
6.43 ± 0.06
|
6.43 ± 0.13
|
NS
|
NS
|
NS
|
T1 = healthy animals on a diet with 32% NDF; T2 = healthy animals on a diet with 36% NDF; T3 = animals which had ERE at the outset on a diet with 32% NDF; and T4 = animals which had ERE at the outset on a diet with 36% NDF. NS denotes values were not significantly different: i.e. P > 0.05. |
The experimental groups were designated as follows: T1 = healthy with 32% NDF; T2 = healthy with 36% NDF; T3 = ERE clinical signs with 32% NDF; and T4 = ERE clinical signs with 36% NDF. The presence of diarrhoea (an ERE clinical sign) was checked by measuring the severity (0 = no diarrhoea, 1 = slight, 2 = moderate, 3 = severe). At the start of the experimental work, all animals categorized as having ERE were around the 1 to 2 boundaries of severity. Information regarding productivity values which were recorded or measured were: initial weight (IW); weekly weight gain; final weight (FW); food consumption (FC) and daily weight gain (DWG).
At 70 days of age, samples of caecotrophs were collected from all rabbits. Collections were achieved by temporarily (07:00 a.m. to 11:00 a.m.) moving rabbits to individual cages and placing polyurethane conical collars (6cm narrow diameter and 27cm wide diameter) on the necks of the rabbits to prevent them ingesting their caecotrophs. Caecotrophs were identified on the basis of their consistency and appearance; they were expelled in the form of clusters with a shiny appearance. During this collection period a sample of faecal waste was also collected, with these being identified as dry and rough, and expelled as individual pellets. The pH of the caecotrophs was measured following collection of 2g samples and mixing with 5 ml of distilled water.
For the determination of SCFA content, 2 g of sample were collected in a 15 ml conical centrifuge tube with 5 ml of HPLC-grade acetone and 1 ml of HPLC-grade water. This was mixed by vortexing for 15 seconds, followed by addition of 100 µl of 85% [w/v] phosphoric acid and the solution was mixed again by vortexing for 10 seconds and centrifuged for 23 min at 21,000 g at 4°C; the supernatant was separated by passing through a 0.20µm filter. The filtrate was subsequently placed in amber vials with a lid to carry out the liquid chromatography analysis and stored at -80°C until used. Liquid chromatography was performed using an Agilent 6890 gas chromatograph with a flame ionization detector and a DB-FFAP 30 m × 0.25 mm × 0.25 µm column (Agilent Technologies, Wilmington, North Carolina, USA) for the analysis of the filtered supernatant with a 0.2 µm syringe filter [19]. Conditions followed a split mode (20:1) with an inlet temperature of 220°C and pressure of 168 kPa. A volume of 1µl was injected, with a constant flow of 1.4 ml/min, using helium as a carrier. The detector temperature was 250°C. The column initially operated at 35°C, with a hold period of 30 sec, before increasing at 10°C/min until 90°C was reached. This temperature was held for 2 min, and was followed by an increase of 12°C/min until 230°C was reached and this was held for 6 min.
For the analysis of ammonium content, a Kjeldahl approach was used [18, 20]. Initially 2 g of caecotrophs were placed in a Kjeldahl flask to which 200 ml of distilled water at 20°C was added, followed by addition of 4 glass boiling beads of 3 mm diameter (PROLAB) and 2 g of MgO. This was then distilled using a recovery rate of 99.5%. The solution was neutralized by addition of 20 ml 0.05 M sulfuric acid for 4 min. The products of the distillation process were collected in 500 ml Erlenmeyer flasks. To each flask 100ml of 2% [w/v] H3BO3 and 3 drops of indicator solution (composed of 20ml of 0.05% [w/v] methyl red and 4ml of 0.2% [w/v] methylene blue) were added to ensure the solution was neutral. Titrations were performed using 0.1M H2SO4 which was dripped with a graduated pipette until the colour of the indicator solution changed. These values were compared against a blank (i.e., without caecotrophs), and the N% of ammonia value was calculated using the equation below:
Equation 1: N% = [(A-B) * 0.014 * N * 100] / M
Where: A and B are the volumes of 0.1M H2SO4 (ml) used in the titration of the caecotroph and control samples respectively, N is the normality of H2SO4 and M is the weight of the caecotroph sample measured in grams.
The data were analysed using a 2x2 ANOVA factorial design, using IBM SPSS Statistics for Windows (Version 19.0. Armonk, NY: IBM Corp). In the case of values which showed significant differences, post hoc analyses were performed.
The rest of the caecotrophs were placed in 1.5 ml tubes which were free of both RNases and DNases and immediately frozen in liquid nitrogen. They were then transported to the Veterinary Microbiology Laboratory of the College of Natural Sciences, Autonomous University of Queretaro, where they were stored at -80°C. DNA was extracted from caecotrophs using Qiagen stool kits with silica and micro pearl columns, according to the manufacturer’s protocol. All caecotrophic and faecal samples (i.e., 16 caecotrophic and 16 faecal samples) were used for sequence analysis.
Next generation DNA sequencing of the V3-V4 region of the 16S rRNA gene was performed on an Illumina MiSeq® System by the Sequencing and Identification Unit, National Institute of Genomic Medicine, Mexico City. The sequencing process consisted of amplicon processing, Ampure beads purification, nucleic acid quantification by fluorescence, automated chip electrophoresis and loading onto the Illumina MiSeq system. The library preparation was performed by the bridge method, with the Nextera ™ Flexible DNA Library Prep Kit (Illumina Inc. USA). Sequences were filtered with Trimmomatic [21] to remove low quality reads, followed by removal of the sequencing adapters, leaving reads of ~ 480bp. A further quality inspection was performed with FastQC [22]: involving trimming; filtering; identifying unique sequences; constructing tables of operational taxonomic units; removing chimeric sequences; assigning taxonomic identification; determining abundance and diversity using the R software Project and the specialized library DADA2 [23]. In addition, PERMANOVA was carried out following the methodology described in the manual of the Vegan package [24]. In the case of the analysis of bacterial diversity this was assessed by calculating the Shannon Index, the Simpson Index, the Chao1 Index and the Abundance-based Coverage Estimators (ACE), following the methodology of Oksanen et al. [24]. The data obtained were compared using a 2x2 factorial design ANOVA using IBM SPSS Statistics for Windows (Version 19.0. Armonk, NY: IBM Corp). A rarefaction curve was produced with the rarefy script: Rarefaction Species Richness in Vegan: Community Ecology Package [24].