PROTOCOLS

Practical, simple, and accurate protocols enable efficient and meaningful research. Amphibian and Reprtile Conservation encourages reseachers to share their protocols through this portal.  

Browne RK. 2014. Formulations for physiological salines for amphibian research. Amphibian and Reptile Conservation. Protocols 1.   PDF

Physiological salines are solutions are formulated to simulate the ionicity – concentration of salts - of various blood and other plasmas, lymphatic fluid, and cellular exudations. In amphibian research they are used for a variety of purposes including standardized aquatic media for oocyte storage, egg incubation and larval rearing, and as bases for cryodiluents. Stock solutions can be prepared and then diluted for use. If stored for any time they should be refrigerated. In veterinary practice moribund amphibians can be placed in tubs with physiological salines to replenish salt loss and reestablish hydration.

Browne RK. 2014. Measuring amphibian morphometrics with ImageJ. Amphibian and Reptile Conservation. Protocols 2.PDF 

These methods use ImageJ™ freeware available at http://rsb.info.nih.gov/ij/ The accurate measurement of amphibian morphometrics is essential for taxonomy, studies of growth and development, and studies of fluctuating asymmetry. The use of digital images and ImageJ™ enables the indefinite archiving of the source image for future reference and gives results that are often more accurate than calliper measurements of struggling animals. Some life stages of amphibians, particularly tadploes, are difficult to measure by hand without risking damage.

Browne RK. 2014. Handling and injecting amphibians. Amphibian and Reptile Conservation. Protocols 3. PDF

There are several methods for the handling of frogs for inspection, injection of medicines or hormones, or passive integrated transponder (PIT) tagging. Generally gloves should always be worn when handling frogs and toads. The wearing of gloves is particularly important for the prevention of disease when handling frogs from different quarantine groups. Some people are allergic to skin secretions - and some frogs have very toxic secretions. Through not wearing gloves there is also the possibility of pathogens including mycobacteria and chlymidea undergoing zoonotic transmission from frog to humans or the reverse.

Browne RK. Figiel CR. Amphibian hormone cycle. Amphibian and Reptile Conservation. Protocols 4. PDF

The amphibian hormone cycle regulates the reproduction of female and male amphibians. The amphibian hormone cycle is quite complex (Figure 1). As amphibians grow to maturity the gonads develop until the testes can produce sperm and the ovaries can produce mature oocytes. Then the amphibian hormone cycle regulates the maturity of oocytes, spermiation in the testes, and the final amplexus and spawning finally leading to the fertilisation of oocytes and the development of eggs.
Mature amphibians don’t spawn until the right environmental cues occur. The mature gonads themselves produce estrodiol that move through the blood stream to the brain. These estrogens regulate the centers in the brain that respond to favourable environmental factors by producing a hormonal cascade through the hypothalamus and then the pituitary.

Browne RK. Figiel CR. 2014. Hormone preparation and use. Amphibian and Reptile Conservation. Protocols 5. PDF

The induction of ovulation of amphibians using hormones has been used since the early 20th century. Some amphibians have never reproduced in captivity without hormones and using hormonal induction many species can be reproduced at will. Conservation breeding programs for the flagship species, the Wyoming toad (Bufo baxteri), Boreal toad (Bufo boreas boreas), Puerto Rican crested toad (Peltophryne lemur), and Chinese giant salamander (Andrias davidianus) are either completely or largely dependent on hormonal induction. Commercial production of African clawed toads (Xenopus spp.) and also numerous other species rely on hormonal induction.

Browne RK. 2014. Optimum egg incubation temperature and pH. Amphibian and Reptile Conservation. Protocols 6. PDF

The optimum temperature at which to incubate amphibian eggs depends on several factors including incubation range, incubation optimum, incubation fluctuation, and production parameters. Important cofactors affecting hatch rate are pH and oxygenation. Oxygen levels should be saturated and pH for most species at neutral of 7.

Browne RK, Antwis R. 2014. Weighing tadpoles. Amphibian and Reptile Conservation. Protocols 7. PDF

Where the variation in tadpole weight within replicate tanks is not required, or where tadpoles are too small to be easily weighed individually, tadpoles can be weighed ‘on mass’ very accurately with the minimum of disturbance or harm to their wellbeing. This technique is commonly used in studies of tadpole growth up until metamorphosis when individual weights must be taken. Front leg emergence is the best stage to measure metamorphosis in anurans. The development of different organs in tadpoles during metamorphosis, and the relative rate of each metamorphosis stage, varies considerably between species.

Browne RK. Seratt J. Counting eggs and larvae. Amphibian and Reptile Conservation. Protocols 8. PDF

Techniques used to count eggs include the „DISPLACEMENT METHOD‟, „IMAGE ANALYSIS‟, and „DIRECT COUNTING‟.