Nitric oxide was delivered to the skin by mixing a gel containing Ascorbic acid (5% w/v) with a gel containing Sodium Nitrite 5% w/v (weight/volume). The Base gel was Klick gel (Klick natural glide, RFSU, Sweden) which has the same composition as the K-Y gel used in the previous publications (Peltonen & Pyornila 2004). When both gels are mixed together over the skin, ascorbic acid rapidly reduces the Sodium Nitrite leading to the release of Nitric Oxide. Equal volumes of both the gels were applied on the skin using sterile cotton bud, mixed well and left for one minute. The gel was then removed gently using a soft tissue.

Three different techniques were utilised to standardize a method to study the brood patch microvasculature.

- Tissue Viability imaging (TiVi)

- Laser Doppler flowmetry (LDF)

- Mediscope imaging