Total bacterial DNA extractions from 500 mg faeces were performed according to Boon et al. [33 (link)]. Isolated DNA was subsequently used as a template to amplify the 16S rDNA for all members of the Bacteria with forward primer P338F-GC and the reverse primer P518r, and a GC-clamp of 40 bp was incorporated into the forward primer. DGGE based on the protocol of Muyer et al. [34 (link)] was performed on the Bio-Rad D gene system (Bio-Rad). The PCR products (10 μL of mixture from 20 μL PCR product and 5 μL loading dye) of the second round were loaded. The obtained DGGE patterns were normalized and analyzed using BioNumerics 2.0 (Applied Maths) [35 (link)]. The number of bands in the DGGE profile was used to calculate the richness in the present study. A matrix of similarities for the densiometric curves of the band patterns was calculated based on the Pearson product-moment correlation coefficient, and dendrograms were created by using Ward linkage [36 (link)].
The quantification of DNA by qPCR was performed with a C1000 Thermal Cycler (Bio-Rad). The amplification and detection were carried out in 96-well plates using SensiMixTM SYBR No-ROX Kit (Bioline Reagents Ltd). Each reaction was done in triplicate in 12 μL total reaction mixture using 2 μL of 50 ng of the DNA sample except for BK where 2 μL of undiluted DNA was used. All qPCR results were expressed as gene copies per g of fresh faeces. The primer sets used in this study are listed in Table2 . A melting curve analysis was done after amplification to confirm specificity of the reaction. Quantification was done by using standard curves made from known concentrations of plasmid DNA containing the respective amplicon for each set of primers.
The quantification of DNA by qPCR was performed with a C1000 Thermal Cycler (Bio-Rad). The amplification and detection were carried out in 96-well plates using SensiMixTM SYBR No-ROX Kit (Bioline Reagents Ltd). Each reaction was done in triplicate in 12 μL total reaction mixture using 2 μL of 50 ng of the DNA sample except for BK where 2 μL of undiluted DNA was used. All qPCR results were expressed as gene copies per g of fresh faeces. The primer sets used in this study are listed in Table
Ingredient composition and nutrient analysis of the experimental diets
| Items | LP | HP |
|---|---|---|
| Ingredients | ||
| As-is basis (g/100 g) | ||
| Pork greaves | 11.0 | 53.3 |
| Brewers rice | 50.9 | 20.0 |
| Lard | 11.0 | 8.50 |
| Rice meal | 15.0 | 6.00 |
| Beet pulp | 3.70 | 5.00 |
| Dicalcium phosphate | 3.00 | 2.10 |
| Yeast | 1.00 | 1.00 |
| Salmon Oil | 1.00 | 1.00 |
| Animal digest1 | 0.89 | 0.89 |
| Calcium carbonate | 0.40 | 0.50 |
| Bentonite clay | 0.50 | 0.50 |
| Salt | 0.81 | 0.42 |
| Vitamin mix | 0.26 | 0.23 |
| Mineral mix | 0.22 | 0.22 |
| Chorine chloride | 0.14 | 0.14 |
| Lecithine | 0.10 | 0.10 |
| Nutrient Analysis | ||
| DM (g/100 g) | 92.7 | 96.2 |
| g/100 g DM | ||
| Ash | 5.35 | 4.75 |
| CP | 17.8 | 50.0 |
| EE | 13.6 | 12.2 |
| CF | 1.05 | 0.91 |
| NFE2 | 62.3 | 32.2 |
| Insoluble fibre | 2.64 | 8.28 |
| Soluble fibre | 2.15 | 0.27 |
| TDF | 4.79 | 8.56 |
| ME, kJ/100gDM3 | 1850 | 1833 |
LP: low protein diet; HP: high protein diet; CP: crude protein; DM: dry matter; EE: ether extract; CF: crude fibre; ME: metabolizable energy; NFE: nitrogen-free extract; TDF: total dietary fibre
1Animal digest: a material which results from chemical and/or enzymatic hydrolysis of clean and undecomposed animal tissue [66 (link)]
2Calculated %NFE = % DM – (% EE+ % CP + % ash + % CF)
3Calculated ME = 16.7×g CP + 37.7×g Fat +16.7×g NFE [27 ]
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