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Frogs were captured by hand at each location; each frog was then weighed and held in a clean container at ambient temperature.
Each frog was then bled via cardiocentesis using 0.5 ml heparinised insulin syringes, and blood samples of 150 µl maximum volume were collected.
The frog was then placed on a foam base that supported the body and the nose in a natural position while leaving the bottom of the mouth freely suspended.
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The frog is then gently massaged then the solution injected is recovered into a sterile tube by puncture with a 22G 1½ gauge needle.
The usually accepted theory to explain these sudden visitations of frogs is that the waters of a pond in which the batrachians were lurking has been sucked up by a whirlwind, that the infant frogs were then drifted in the clouds and came down when the storm broke.
In this modified tree, humans are still sister to cats and birds are sister to lizards, frogs are then sister to amniotes, and fishes are the outgroup to the tetrapods.
The frogs were then wrapped in moist gauze to prevent dehydration and allowed to recover fully from anesthesia.
The frogs were then rinsed in fresh water until conscious, and placed in their original container until all signs of anaesthetic exposure had passed for at least 10 minutes.
Testosterone levels from the experimental frogs were then determined from the standard curve.
Frogs were then swabbed on their dorsal and ventral surfaces separately using sterile swabs (Eurotubo, Rubi, Spain).
Frogs were then incubated in 100ml of water containing 1mg/ml bromodeoxyuridine (BrdU, Sigma St . Louis MO; Catalog #B5002-1G) 2 days before sacrifice.
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