Does mitochondria function control pigmentation in male Zebra Finches?

During the 21 days of treatment the birds will be housed in individual 24”x16”x16” enclosures divided in half length-wise with a removable wire divider with ad libitum access to food and water. Birds will be left untouched for at least 24 hours after placement in these cages to allow them to adjust to their setting. For the experimental group, Mazuri ® food pellets will be supplemented with 625 ppm DNP per day following the work of Dominguez et al. (1993) and Stier et al. (2014) in which mitochondria membrane dysfunction was induced in small birds with minimal stress on the animal. The control group will be fed food with only the propylene glycol solvent. One key difference between our protocol and the work done by Stier et al. is that our DNP will be supplemented in the food instead of the finch water supply. This should allow us greater control over the amount of DNP ingested by the birds than in the previous work. Although Zebra Finches must be allowed to feed ad libitum, we can measure by weight the amount of food being eaten every 48 hours in order to assure the birds are 1) eating the recommended amount to maintain body weight and health and 2) taking in the correct dosage of DNP (Dominguez 1993). Fresh food with supplement will be replaced daily along with the water supply to ensure proper nutrition.

After treatment, each bird from both the experimental and control groups will have the carotenoid coloration of their bill and legs analyzed. Assessing the quality of red coloration using photography and color standards is a straightforward process and is key to describing the quality of the overall mating display (McKay 2013). We will adapt the suggestions and techniques used by Stevens et al. (2007).  Zebra finches will be gently restrained by hand and held in front of a Kodak ® 18% gray card background. Images will be taken with a professional Nikon ® camera in RAW format and saved as a TIFF file to ensure there is no compression or distortion of color pixels. The color of the Zebra Finch beaks and legs will be compared to high quality Kodak ® color standards to determine color change from before to after treatment.

Similarly, spectral data measured using a portable spectrometer will be used to measure the difference in carotenoid reflectance between the experimental and control groups after the treatment according to procedures adapted from Macedonia et al. (2000) and Toral et al. (2008). Measuring reflectance will provide important data on the intensity of color reflected by the carotenoid displays in male birds. Each bird will be held per the same method of gentle restraint and placed in front of a dark black background in order to assure maximum reflectance from the beak and leg coloration reaches the sensor probe of the spectrometer. The sensor probe will briefly be placed at approximately a 70º-90º angle close to, but not touching, the bird. Measurements will be taken in triplicate (to account for variations in movement) using a light source with known characteristics.  A flash will NOT be used to avoid damage the retina of the Zebra Finch or cause disorientation.

Following the conclusion of the three weeks of treatment, and once photographical and spectral data has been recorded, the birds will be sacrificed according to the humane methods in order to obtain carotenoid containing integuments from the beak and leg tissues for HPLC analysis. Extraction of carotenoid pigments from the beaks and leg integument and subsequent HPLC will be performed according the procedure established by Mundy et al. (2016). Sacrifice of the birds for this step is necessary as it is not possible to biopsy tissues from the beak or legs without causing unneeded pain for the animals. Additionally, HPLC is the most current, advanced, and accurate method for determining carotenoid concentration in the tissues of living organisms.

The liver of each bird will also be extracted to isolate mitochondria from the hepatic tissues. The respiratory control rate (RCR) of the mitochondria will be recorded based on established methods by experienced researchers Dr. Tonia Schwartz (Schwartz and Bronikowski 2013, Schwartz et al. 2015) and Dr. Andreas Kavazis (Kavazis et al. 2009). This is a necessary step because it will allow us to demonstrate that mitochondria function has been disrupted (Brand and Nichols 2011).

It is important to note that through each step of the experimental protocol, every effort must be taken to avoid adding stress to the birds. This is not just for the sake of the animal even though the well-being of the birds is of the upmost importance. It is also suggested that added stress from natural, environmental challenges such as bacterial infection may lead to decreased carotenoid production in birds (Brawner et al. 2000, Hill et al. 2004). Therefore, it also benefits the investigator to avoid any stressor to the birds to control confounding variables that could affect the red color production in the zebra finch.

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