*3.5.1. Total RNA extraction and reverse transcription*

Approximately 30mg of macro-dissected caecal tonsil tissue per sample (previously submerged in RNAlater® (Sigma-Aldrich, USA) and stored frozen at -80°C) was homogenised in 500 uL QIAzol lysis reagent for 10 min at 30 Hz in a Tissuelyzer LT (Qiagen, UK). Lysates were mixed with 100 μL chloroform, transferred to pegGold PhaseTrap tubes (PeqLab, UK) and centrifuged for 5 mins at room temperature. The aqueous phase was poured into fresh tubes, mixed with 1.5 volumes of ethanol and applied to Qiagen RNeasy columns (Qiagen, UK). RNA was purified according to the manufacturer's instructions (Qiagen, UK). RNA integrity was assessed using an Agilent Bioanalyzer and RIN was >8 for all samples. Purity and quantity were measured using a NanoDrop spectrophotometer; for all samples the absorbance peak was at 260 nm, A260/280 > 2 and A260/230 > 1. About 800 ng of RNA were reverse transcribed using a Quantitect reverse transcription kit (Qiagen, UK) in a 10 μL reaction according to the manufacturer's instructions. This RT kit includes a mandatory gDNA wipe out step. The completed reaction was diluted 10-fold with 5 μg/mL tRNA in water.

for product specificity (single peak) and the presence of primer dimers. All primers were designed to be intron-spanning so that any residual gDNA present could not be detected and avoided known SNP and secondary structure. Assays were designed by qStandard (www. qstandard.co.uk) and were tested for specificity by electrophoresis, efficiency >95%, sensitivity to 10 copies/rxn, and linearity over 7 log by qPCR. Copy numbers/reaction were derived from the standard curves using the Rotor-Gene software. The four reference genes identified as the most stable using geNorm software were B2M, GAPDH, PPIA and YWHAZ. The normalisation factor for each sample was used to normalise GOI copy numbers per reaction.

Plant Extracts, Energy, and Immune Modulation in Broilers

http://dx.doi.org/10.5772/intechopen.77220

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Data were statistically analysed by two way analysis of variance (ANOVA) using a 2 × 2 factorial arrangement of treatments, blocked by experiment. The main effects were the cereals (maize and wheat) and additives (with and without PE). All data were processed using the procedure of Genstat (18th Edition) statistical software (IACR, Rothamstead, Hertfordshire, UK). In all instances, differences were reported as significant at P < 0.05. Graphics (**Figure 2**)

Dietary PE supplementation significantly improved (P < 0.05) gain to feed (G:F) ratio by 2 points and dietary NE by 0.34 MJ (**Table 2**). No changes (P > 0.05) were observed in dietary ME due to PE supplementation. The increase in feed efficiency is in agreement with the ability of spices and mixtures of spices to increase bile secretion, activity of the pancreatic, and brush border enzymes [43, 44]. Maize based diets produced higher (P < 0.05) daily FI and ME, although wheat based diets had higher NE (P < 0.001). The values of ME and NE were in similar to previous reports [9, 45]. In agreement with [24], there were dietary type x PE interactions (P < 0.05) observed in bird growth, as birds fed wheat diets did not respond (P > 0.05) to PE supplementation. Similar tendency (P = 0.074) was observed for daily feed intake. Compared to maize, wheat contains more water-soluble non-starch polysaccharide (NSP), a carbohydrate complex possessing antinutrient activity, which may reduce dietary nutrient availability [46], thus explaining the reduced performance of birds fed wheat based diets. The observed interaction may also be due to the relatively high fat content of the wheat compared to maize based diets, and not to the cereals alone. Widening the dietary ME to protein ratio is likely to affect body fat retention more than bird growth performance, suggesting an explanation for the inconsistency between weight gain and NE of birds fed wheat based diets. However, the impact of dietary formulation (cereals, protein sources, fat content etc.) on the effectiveness of supplementary PE in poultry nutrition warrants further investigation. Although there is a lack of consistency between growth performance and dietary ME, this is in agreement with many studies [8, 10, 11] but is in disagreement with others [9]. The

were produced in "ggplot2" package version 2.2.1. [41] using R version 3.4.1. [42].

**4. Effect of PE on bird growth performance and dietary available** 

**3.6. Statistical analysis of data**

**energy**

#### *3.5.2. Quantitative real-time PCR*

Two microlitres of cDNA were amplified in a 10 μL reaction using Agilent Brilliant III SYBR Ultra-Fast SYBR Green mix with each primer at a final concentration of 500 nmol/L. The notemplate control reaction contained 2 μL of tRNA (0.5 μg/mL). DNA standards (10^7–10^1 copies/rxn) for each gene were included in each run. Reactions were pipetted robotically using a Qiagility (Qiagen, UK). Amplification parameters were: 95°C for 3 min followed by 40 cycles of 95°C for 5 sec, 57°C for 1 sec in a Rotor-Gene 6000. Melt curves were checked

**Figure 2.** The effect of dietary plant extracts (PE) on the normalised mRNA copy number (per reaction) of (a) CD40 LG, (b) IL-12B, (c) INFG, (d) IL-6 in chicken caecal tonsils. Error bars represent ±1 pooled SEM.

for product specificity (single peak) and the presence of primer dimers. All primers were designed to be intron-spanning so that any residual gDNA present could not be detected and avoided known SNP and secondary structure. Assays were designed by qStandard (www. qstandard.co.uk) and were tested for specificity by electrophoresis, efficiency >95%, sensitivity to 10 copies/rxn, and linearity over 7 log by qPCR. Copy numbers/reaction were derived from the standard curves using the Rotor-Gene software. The four reference genes identified as the most stable using geNorm software were B2M, GAPDH, PPIA and YWHAZ. The normalisation factor for each sample was used to normalise GOI copy numbers per reaction.
