QUINTA UNIDAD DIDÁCTICA: (40 HORAS)

Individual pigs for blood sampling and euthanasia were selected to be closest to the mean average weight for the pen replicate. This is reflected by the data in Table 6.12 which confirms they were consistent with the growth rates in Table 6.11. Systemic WBCs are markers of immune system activation and while Zn has not been shown to affect activated neutrophils and IFN-γ producing cells post-weaning (Jenson-Waern et al, 1998, Chai et al, 2014), little is known about the influence of phytase. The effects of high doses of phytase and ZnO are more pronounced in the first two weeks post-weaning, so blood analysis to assess general health status was conducted at 12 days post-weaning. Analysis revealed no effect on total WBC (Table 6.13), with levels within the normal biological range expected (Kahn et al., 2005). There is therefore no clinical relevance of this data, which is to be expected since the pigs were not challenged in this experiment.

In the group fed the combination of additives, circulating lymphocyte percentage tended to be higher than when phytase and ZnO were either not fed or included on their own (Figure

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6.2). This group also had significantly higher expression of the pro-inflammatory cytokine interferon-γ in the duodenum compared to the control group, suggesting intestinal inflammation in the anterior section. This was not detectable in the ileum, possibly related to a reduced buffering capacity at the stomach/duodenal junction or differences in the upper small intestine microbiome (Starke et al., 2014). The addition of 100 mg/kg CS-Zn and 500 mg/kg ZnO have been shown to rival doses of 3000 mg/kg and 2250 mg/kg ZnO respectively for improved small intestine histophysiology and immunity (Han et al., 2014; Hu et al., 2013). In this study, both the control group and the phytase group diets contained 150 mg/kg Zn. This may have limited the detection of differences expected from the high Zn treatment as the basal level of zinc was probably sufficient for optimal gut health and growth. Pharmaceutical ZnO supplementation alone did not significantly affect TNF and IFN-γ mRNA abundance in this study either and expression levels of both cytokines relative to the control were low (<0.5 log10 change), suggesting no major challenge was taking place

at day 21 post-weaning. Overexpression of pro-inflammatory cytokines have been implicated in reduced tight junction protein expression (Hu et al, 2013), thereby leading to reduce intestinal integrity.

Many studies rely on only one reference gene (RG) for normalisation of the transcript expression of genes of interest (GOI). Often the gene selected has not been assessed for stability or suitability and is chosen entirely for historical reasons. Normalising GOI expression in this way is prone to error, as using a single RG has been shown to vary greatly in stability depending on tissue type (Ryan et al., 2010) and should not be used without validation (Bustin et al., 2009). Prior to the GOI expression assays, a geNorm™ experiment was conducted to find appropriate reference genes to normalise the cytokine data (see section 3.6.2.). The results showed that the best combination in duodenum tissue was GPI and GSR, so these were subsequently used to normalise the GOI data. To improve the accuracy of the Cq values, PCR efficiency was estimated using regression analysis on a four-point-window of linearity of the log-linear phase, by amplicon group (Ramakers et al., 2003; Ruijter et al., 2009). The NRQ presented in Figures 6.3 and 6.4 are therefore likely to be as accurate as currently possible. The addition of no template controls and reverse transcription negatives also assisted in stringent quality control. As a consequence, the IL- 6 assay failed the QC by amplifying gDNA (despite a DNase step and a small intron being present, the assay was found to “jump”) and the results were therefore not included in this chapter. The one pig that was found to have a bladder infection upon post-mortem inspection showed highly elevated TNF levels in the intestine and was also excluded from the data analysis.

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6.6. Conclusions

In conclusion, the addition of super dosing phytase (2500 FTU/kg feed) was confirmed to be efficacious at improving production performance (8 % increase in DLWG and 0.07 point decrease in FCR) if fed up to 14 days post-weaning. In this study with pigs showing no clinical signs of PWD, medicating feed with pharmaceutical ZnO (3100 mg/kg feed) is contraindicated, as it was shown to significantly reduce production performance in this chapter, especially in diets containing relatively low levels of digestible phosphorous. Where pharmaceutical ZnO is included for seemingly healthy pigs, this should not be in conjunction with super doses of phytase, due to potential inhibition of phytase activity. Higher levels of dgP from inorganic sources of between 1 – 2 g/kg more than normal may be warranted for optimal growth response in ZnO medicated diets. In any case, supplementation with pharmaceutical ZnO should not exceed 14 days post-weaning.

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CHAPTER SEVEN

General discussion and conclusions on super dosing phytase

and supplementation of pharmaceutical zinc oxide for enhancing

the production performance of newly weaned pig

7.0. Introduction

Pharmaceutical grade ZnO is a widely used replacement to antibiotic growth promoters in many EU countries (EC, 2015). There is, however, growing concern about the environmental impact of increased zinc usage in the pig industry (SCAN, 2003), lack of consistent efficacy and possible development of microbial resistance (Slifierz et al., 2015) developing. Research into alternative growth promoters and their implications for use in ZnO medicated feeds is therefore of growing interest to the pig industry. A new strategy for using phytase enzymes by super dosing at levels beyond that generally required for improving P digestibility had emerged as a possible growth promoter but direct evidence of ‘extra phosphoric’ effects in newly weaned pigs was not well documented. The objective of this thesis was therefore to evaluate the efficacy and possible explanatory mechanisms of super dosing phytase at enhancing pig performance post-weaning, beyond that attributable to increased phosphorus digestibility. In addition, the thesis has assessed the suitability for super dosing phytase in ZnO medicated feed.