APSS 2013 Proceedings - The University of Sydney

Aust. Poult. Sci. Symp. 2013.....24
Broiler premix (d5 – wk 11) and Alltech PN Pre-Harvest Broiler premix modified to contain
2.0 g/kg available P and 7.0 g/kg Ca (wk 11 – 17, Conditioning-PN).
At 17 weeks of age, eight birds from each treatment were randomly selected and
euthanized by argon asphyxiation followed by cervical dislocation. The small intestine was
quickly removed, rinsed to remove contents and a sample from jejunum section 3cm
proximal to Meckel's diverticulum was flash frozen in liquid nitrogen and stored at -80°C
until further analysis. Total RNA was isolated from the jejunum using the RNeasy Mini Kit
(Qiagen) according to the manufacturer’s suggested protocol. Total RNA (0.5 μg) was
reverse transcribed into cDNA using the High Capacity cDNA Reverse Transcription kit
(Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions.
Real-time PCR was performed in triplicate using Taqman Gene Expression Assays (Applied
Biosystems) and the 7500 Real-time PCR system (Applied Biosystems). Ring finger protein
4 (RNF4) was selected as an endogenous control gene to account for any variation in the
efficiency of reverse transcription and PCR. The relative quantification (RQ) was expressed
as a ratio of the target gene to control gene using the delta-delta Ct ( ΔΔ Ct) method. Data
presented are least square means (lsmeans) ±SEM. Real-time PCR results for Normal-PN
and Conditioning-PN were compared with ANOVA analysis for a completely randomized
experimental design using the Proc GLM procedure of the SAS version 9.1 statistical
package (SAS Inst. Inc., Cary, NC, USA). Comparisons of the treatments to the control were
made using a Student’s t-test.
III. RESULTS AND DISCUSSION
Relative expression of zinc transport protein (ZnT) 1 and 7 did not differ between treatments
(Figure 1). Expression of ZnT4 was increased in both Conditioning-PN- and Normal-PN-fed
birds than control (P < 0.05), but did not differ between birds fed the Conditioning-PN and
Normal-PN treatments. Normal-PN fed birds had greater ZnT5 and ZnT6 mRNA levels
compared with control (P < 0.05), but levels did not differ from the Conditioning-PN-fed
birds. Relative mRNA levels of ferritin, heavy polypeptide 1 (FTH1), metallothionein 3
(MT3) and metallothionein 4 (MT4) did not differ between treatments (Figure 2). The posthatch
conditioning supplement had no effect on calbindin 1(CALB1) mRNA levels, however,
expression was greater in both Conditioning-PN- and Normal-PN-fed birds than control at 17
weeks of age (P < 0.05). Calbindin 1 levels track directly with changes in Ca absorption with
a correlation coefficient of 0.99 (Morrissey and Wasserman, 1971). Therefore birds fed
either treatment in this study would likely result in an increase in Ca absorption. Somewhat
surprisingly, solute carrier family 34 (sodium phosphate) member 2 (NaPi-IIb) mRNA levels
were reduced 1.37-fold (P = 0.03) in Conditioning-PN birds compared with the control.
When dietary P levels are reduced, there is an adaptive response in which NaPi-IIb mRNA
abundance increases (Giral et al., 2009), therefore it would be expected that NaPi-IIb mRNA
abundance would be greatest in both the Conditioning-PN- and Normal-PN-fed birds where
available P levels were reduced in the starter and grower phases. This may also indicate that
P release by the enzyme inclusion, which among others provides 300 SPU/kg of phytase
activity, in the diets containing Alltech PN premix resulted in the down regulation of NaPi-2b
to regulate P uptake when more P was available.
IV. CONCLUSION
Results from this study indicate that, in layers, the expression of some nutrient carrier and
transport genes can be influenced by a post-hatch preconditioning period. Research from this
lab has shown that diets fed in early life (4 d post-hatch) can have lifelong effects on
expression patterns of nutrient carriers and transporters in the intestine regardless of diets fed
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