MALE	FEMALE
Weight range	27 to 54g	43-140g
Body length range	6.1-7.5cm	8.1-8.4cm
Head length average	1.8cm	2.7cm
Head length range	1.8-1.9cm	2.2-2.9cm
Head width average	1.8cm	2.5cm
Head width range	1.8-2.0cm	2.5-2.6cm
TOTAL LENGTH	HEAD LENGTH	HEAD WIDTH
Snout to Vent	Snout to middle of head at the end of cranial crest	Between the posterior edge of the cranial crests

When the males are in prime breeding condition, sexual dichromatism is particularly evident. Their color changes to lemon yellow, on the sides of the body and throat area. Males also possess prominent nuptial pads on the first and second digits of the front feet. Males will produce a male release call. By clasping the toad just behind its front legs, with thumb and forefinger, it is possible to elicit a chirping release call from a male. This chirp may be barely audible or quite loud, depending on the toad, but in any case it is often possible to feel the jerking of the male's body as he exhales air to produce the call. Although this method is not infallible, as some males do not readily sound the release call and some females will attempt to do so.

Conditioning for Breeding:
During the dry months in Puerto Rico, crested toads become dormant and aestivate. This rest period may act as a stimulus for egg maturation in the ovaries of females. The conditioning period prior to breeding Puerto Rican crested toads appears to be about 30-45 days. In the past, the pre-breeding conditioning involved aestivating toads in a moistened substrate of sphagnum and peat moss at a maintenance room temperature of 30oC. The substrate was allowed to slowly dry out, but it was found that the toads dehydrated as well. This method was discontinued due to toad deaths during and post aestivation.

Egg production and maturation in captive amphibians has often been unpredictable, possibly due to abnormalities in the vitellogenic cycle. Keeping amphibians at lower temperatures for 4 to 6 weeks has been shown to partially correct abnormalities in the vitellogenic cycle of captive amphibians. These animals produce larger numbers of mature ova that can support development. Through the limbic system, environmental cues such as temperature and light may affect the hypothalamus, which secretes gonadotropin-releasing hormone (GnRH). GnRH causes the pituitary to release gonadotropin, which in turn stimulates steroid hormone production. The oestrogen steroid hormones play an important role in stimulating yolk protein synthesis in most vertebrates.

Toads are typically maintained at a temperature range of 26oC-30oC (78oF-86oF). For pre-breeding conditioning, the SSP recommends that female toads (studies at Toronto Zoo have shown that it is not necessary to cool males) be cooled for 30 days. Before this conditioning period, toads are weighed and a general health examination is performed. If fat stores appear good and weight has remained stable, the selected toads are prepared for hibernation. The toads are not fed two weeks prior to and during the cooling period, but always have access to water. When a large number of toads are to be bred, the room in which the toads are housed may be cooled. For smaller numbers, the toads are moved in their existing tanks, as is (i.e. no substrate other than the rubber matting and hides), into an environmental chamber or incubator for cooling. The temperature is slowly dropped to 18oC (66oF) over the first 5-7 days of the cooling period. The open mesh of the aquarium lid is partly covered to raise the humidity to over 80% in the aquarium. As little disturbance as possible is encouraged during the cooling period.

To reduce the possibility of introducing chytridiomycosis to Puerto Rico, a 5-day prophylactic anti fungal treatment (0.01% suspension of Itraconozole) is used for all toads recommended to breed with offspring destined for release. (Note: chytridiomycosis has not been documented in crested toads, but it has been diagnosed in other bufonids.) Females receive their treatment prior to cooling and the males receive theirs prior to being placed with the females. It is also recommended that a rinse spray of Itraconozole be applied to the contents of the breeding tank before toads are introduced.

Breeding:
Captive breeding events are scheduled for late fall (Oct-Nov) to coincide with the likelihood of rain in Puerto Rico. If an important fall breeding fails, then an early spring (March-April) breeding may be attempted. It is important to conduct a fecal examination after each breeding attempt. Despite normal loads, the stress of conditioning may affect the immune system response, and bacteria/parasite loads may be elevated.

Breeding tanks are set up with aged tap water a minimum of 14 days before the pre-breeding conditioning period begins. This allows for adequate preparation of filters and allows sufficient algae growth for tadpoles to feed on. Tanks range in size from 20 gallon to 100 gallon glass tanks or plastic tubs. Breeding tanks can be set up on a slant with approximately 8cm(3") of aged or dechlorinated water at one end and a deeper end with 15cm(6") of water, or rocks can be strategically placed within a tank to prevent drowning and allow the toads choice of water depth. The tanks are seeded with algae for tadpole food. The water temperature is maintained at 25.5 C (78 F0 to 30oC (86oF). Large tubs can be set up with sponge filters and a canister filter circulating the water and a return that is pumped through a perforated wand in an effort to simulate rain. Smaller aquaria should also be equipped with some form of mechanical filtration (sponge filter, magnum, etc.).

A calcium block, used in home aquariums, can be placed in tanks. Plastic plants, rocks and large half clay pots, some submerged, others half submerged to form out of water perching spots, are placed throughout the tank. Algae grows well on these and adults string their eggs around the clay shards and the plastic plants. In the wild, emergent plants are important as egg laying sites.

At the end of the cooling period the toads are warmed back up to 28oC (82 oF) over three days (i.e. approx. 3 oC per day). Day 1 marks the first increase in temperature. The toads are not otherwise disturbed on this day. On day 2 the tanks are filled with 1” of dechlorinated aged water. Taped toad calls are played throughout the day and into the night. As early as possible on the morning of day 3 the males are placed into the large breeding tank with simulated rain and the taped calls are played throughout the day. Approximately 6 hours later in the afternoon of day 3, the females are added to the breeding tanks. Wrestling of competing males appears to be very stimulating to male toads. As toads amplex they are moved to a separate breeding tank to keep track of parentage. If toads do not amplex, hormones are used (see protocol below). Eggs are usually laid overnight within 24 hours of amplexus or after hormonal injections. Once eggs are laid, the adults are removed to holding tanks and the eggs are allowed to hatch in-situ. The water level is then gradually increased in order to provide a larger volume of water for developing eggs.

Hormone Induction Protocol:
The following protocol is used for the induction of reproduction in Puerto Rican crested toads at SSP facilities has proven successful on most occasions. The most success has occurred in young toads in their first breeding season, (males 1-2 years old and females 2-3 years old) however, hormone injections have stimulated egg laying in 7-year-old toads at the Cincinnati Zoo, and 9-year-old toads at the Toronto Zoo. The use of hormones for breeding amphibians does not negate proper pre-breeding conditioning. Hormone treatment will not be effective in the absence of mature ova in the ovary. In other anurans, ova maturation is dependent upon an extended maturation time and a good nutritional status. Stress is a powerful inhibitor of amphibian reproduction. It has not been possible to induce ovulation in Puerto Rican crested toads that have not been through a cooling period. In some cases, pairs of toads will go into amplexus for variable periods, but fail to deposit eggs.

The luteinizing hormone releasing hormone analog des-Gly (d-Ala) LHRH ethylamide from Sigma Chemical (catalogue number L4513) is used. Sigma Chemical now requires USDA approval to sell LHRH for use in amphibians. The product comes as a powder in 1 or 5 mg bottles, needs to be dissolved prior to use, and should be stored in the freezer. At TZ, LHRH is diluted to make a 100 microgram/ml solution and frozen in 1 ml syringes prior to use. The shelf life of the dry product is 2 years. Whenever possible, new product should be obtained for each breeding cycle.

If toads are in amplexus, females are injected using a dose of 0.1 ug/g subcutaneously. Intraperitoneal injection can also be used. These doses are higher than those used by some working with other species, but each species can be slightly different and there has not been enough research to find the minimum or the optimum regime. If no eggs are laid overnight, then females may be injected again the following day. As a general rule, if a hormone injection does not work the first time, it is not likely to work later. However, toads may be injected up to three days in a row with no known adverse affects. Memphis Zoo is currently conducting research on the use of hormones in Bufonids to improve artificial reproduction techniques. It is worthy to note that some females seem to be susceptible to fluid overload during or shortly after the breeding process and death from drowning can occur from overzealous males.

If pairs are not in amplexus, injecting females may cause premature deposition of unfertilized eggs. In these cases, males are injected first to induce amplexus behaviour and insure females do not “dump” their eggs without being fertilized. Females in amplexus are subsequently injected once a male has amplexed with them. Further injections are usually not required for males. However, if they do not remain on the females, multiple injections may be warranted.

Egg, Tadpole and Toadlet Husbandry:
After the adult toads are removed from the breeding tanks and detritus is siphoned out, care is taken not to disturb egg strands. Filters and return water are moved away from any eggs. Water depth is slowly increased until it reaches 18-23 cm (7-9"). Egg development is rapid at 30oC (86 F) and development is seen within 12 hours. Normally after twenty-four hours, the eggs hatch and the embryonic tadpoles cling to the egg strings before dropping to the bottom of the tank. It is important not to disturb the embryonic, externally gilled tadpoles at this stage. The tanks are left another 24 hours until the gills are absorbed and the tadpoles begin to swim. Any unhatched eggs are then carefully siphoned out of the tank. Aeration by the airstone or filter is kept low to avoid a whirlpool effect on the eggs and newly hatched tadpoles. It is necessary to cover the filters or overflows with a fine netting (panty hose works well) to avoid trapping the tadpoles inside of the filter.

To maintain the highest water quality it is advisable to monitor water quality on a regular basis due to the presence of excessive bioloads. Detritus and uneaten food need to be siphoned off and measured water changes with dechlorinated water (up to 40%) should be conducted when ammonia levels are high. Successful metamorphosis is more likely if the tadpoles are left in large volume tubs. When food items are placed in the tanks, a few tadpoles begin to feed, starting what appears to be a swarming around the food items. Tadpoles appear to move en masse throughout the tank.

The tadpoles are fed three times daily so that old food can be removed and fresh food is always available. The morning feeding consists of equal parts Sera-san enhancing flakes, Tetra 4 in 1 FD menu, aquaria herbivore diet, and spirulina flakes. The food is moistened with a few drops of water to form a firm ball and rolled in ascorbic acid powder. For example, 1/4 teaspoon (approx. 0.6 grams) of food plus supplement feeds 50 tadpoles (10 days old) at one feeding. Three to four feeding stations are provided per tank. The small balls of food are stuck to the tank wall just at the water surface, or are allowed to fall to the bottom of the tanks. The amounts and number of feeding stations are increased as the tadpoles grow. Calcium blocks will slowly dissolve in the water. A new one should be added to the water before the old one has completely dissolved.

Food from the morning feeding is removed in the afternoon prior to the second feeding. The afternoon feeding consists of Sera-viformo tablets. Two tablets (approx. 0.5 grams) are given per 50 tadpoles (at 10 days old).

The third feeding consists of pieces of 4" by 4" frozen-thawed or microwaved (to rupture the plant cells) spinach or lettuce per tank. Lettuce and spinach are fed on alternate days and the pieces are left in the tanks overnight. This provides an overnight food source with little risk of fouling the water.

Prior to the morning feeding, the remaining food and fecal material from the previous night are removed. The pH, ammonia levels and the water temperatures are checked. Ammonia levels under 0.1 ppm, nitrates below 1ppm, pH between 7 and 8, and temperature of 30 oC (86oF) are recommended. Water changes are performed if needed. Water temperatures have ranged from 21-34oC (72-93oF) over the course of tadpole development. Recent studies at Toronto Zoo suggest that a temperature of 30oC (86oF) is best suited for raising healthy tadpoles. Ammonia and pH levels have ranged from 0.02 to 3.3 ppm and from 6.7 to 8.3 respectively, prior to morning water changes. Daily water changes vary from 10% to 50% depending on pH and ammonia levels and the clarity of the water. Sponge filters are rinsed clean with dechlorinated water once per week; filter wool and activated charcoal are changed three days later. Sudden fouling of the water due to tadpole mortalities or overfeeding can be addressed with massive water changes of up to 75% using fully aged or dechlorinated water.

From studies done in Puerto Rico, metamorphosis occurs approximately 21 days after hatching. At cooler temperatures, captive metamorphosis can occur 10-14 days later than wild tadpoles. At 30oC (86 F), hind legs are obvious after day 9 and some tadpoles are ready to metamorphose by day 14. At this point "beach" areas are created in the tanks, by slowly dropping the water levels and offering more plant material and cork bark. From day 10 to day 21 (some tadpoles are delayed in development and some fail to metamorphose) front legs appear and toadlets start appearing on the "beaches."

As toadlets start appearing on the "beaches," they are moved to toadlet rearing tanks. Water is added to increase humidity and to prevent dehydration. The tank is set up with rubber matting, indoor/outdoor carpeting, or gravel at one end of the tank and half coconut shells, clay shards, plastic hides, plastic plants and leaves are offered as refuges. One cm (.5 inch) of water is offered at the other end. To avoid desiccation after metamorphosing, the toadlets seek cover as soon as they leave the water. Although many hide spots are offered, toadlets usually mass together in large numbers under one or two hides. This may be an adaptive behaviour for moisture conservation since the surface area to mass ratio is reduced. This drought reducing behaviour has been observed in both captive and wild situations with captive-bred toadlets.

Newly emerged toadlets are quite capable of scaling vertical glass walls of tanks, so secure lids are required. Lids should have sections screened for good air circulation. This also allows for some control over humidity levels, increasing or decreasing it by covering or exposing the screened areas of the tank lids. Since young toadlets are susceptible to dehydration, humidity in the tanks should be maintained over 80%.

The main food items for toadlets are pinhead crickets. If gravel is used as a substrate, care is required to ensure that the small crickets do not hide or disappear into the substrate. Fruit flies may be fed as well in the first few weeks. The appropriate amount of food to offer for a group of toadlets can be determined by using Graph 1 below. (For example, a group of 25 toadlets at 5 weeks would require 2.5grams of food.)

As the toadlets increase in size, so does the cricket size. At eight to ten weeks they are offered 1/4" crickets, at 16 weeks they are offered 1/2" crickets and at 24 weeks they are offered 3/4" adult crickets. At 18 weeks the toads are reduced to four feedings per week. As they approach adult size, other food items such as wax worms or newborn mouse pinkies are introduced. At this time they are put on the same feeding schedule as adults and the amount of crickets offered is slightly reduced.

Reference:
Lentini, A. 2000. Puerto Rican Crested Toad (Peltophryne lemur) SSP Husbandry Manual. Keeper and Curator Edition. Toronto Zoo: Scarborough, Ontario. 48pp.