Analysis five

All analyses were performed on coded sections using AnalySIS FIVE (Olympus Soft Imaging Solutions, Münster, Germany) following the suggestions for standardized nomenclature from the American Society for Bone and Mineral Research [24 (link)].

For blood smears screening, examination of sporogonic stages and parasitaemia calculation in experimental individuals an Olympus CH2O light microscope with × 40 and × 100 magnifications was used. Pictures for measurements of the parasite at its different blood stages were prepared by using the Olympus BX61 light microscope equipped with digital camera DP70. Visualization of pictures was performed using the software AnalySIS FIVE (Olympus Soft Imaging Solutions GmbH, Münster, Germany). Blood smears obtained from wild birds were examined about 15–20 min with × 100 magnification. To evaluate the intensity of parasitaemia, numbers of infected erythrocytes per 10,000 red blood cells were counted [28 (link), 31 (link)].

Fields within stained cross‐sections were captured at 40 × magnification using a microscope with a digital camera (BX60; Olympus Optical, Ltd., Tokyo, Japan). A total of 4 sample fields were selected from all stained sections in each muscle to account for non‐uniform fiber type distribution. For example, in muscles with significant fiber type compartmentalization (such as MG and TA), the deep and middle parts were randomly sampled. Muscle fiber type and muscle fiber cross‐sectional area (CSA) were measured in approximately 200 muscle fibers from each muscle in each animal. The same fibers selected for CSA measurement were used to determine fiber type proportion in each muscle. Muscle fiber type was distinguished according to the staining intensity from the histological procedure described above (I > IIX > IIB > IIA, dark to light; Fig. 2). A blinded assessor performed all the muscle measurements manually using the AnalySIS Five software (Olympus Soft Imaging Solutions GmbH, Hamburg, Germany).