Eggshell Thickness
SEM (FEI Quanta 200 ESEM, Eindhoven, Netherlands) was used to observe
the thickness of the eggshell, the thickness of each constituent layer
and external features. The samples were rinsed with reverse osmosis
water to further remove any particles and allowed to air dry; samples
were then mounted on aluminium stubs and gold spluttered with
approximately 100 nm of gold in a vacuum (Baltec SCD 050 sputter
coater). The images were taken by the Manawatu Microscopy and Imaging
Centre (MMIC), using an accelerating voltage of 20kV and a spot size of
3 to 4. For this technique, eggshells of 23 Brown Kiwi, 4 Roroa, 21 Rowi
and 16 Haast Tokoeka were used.
Images of the cuticle were resolved at 20 μm resolution, cross sectional
images and internal side images were produced at 300 μm. The
measurements were analysed using ImageJ free software (Rueden et al.
2017).