Action: Use artificial fertilization in captive breeding
- Three replicated studies (including two randomized studies) in Australia and the USA found that the success of artificial fertilization depended on the type and number of doses of hormones used to stimulate egg production. One replicated study in Australia found that 55% of eggs were fertilized artificially, but soon died.
Reproductive technologies such as artificial fertilization are techniques that can be used in an attempt to achieve or increase breeding success by captive amphibians. Many amphibians have external fertilization, making this technique relatively simple. However, consideration must be given to the storage and ratio of sperm and eggs, effects of temperature, solution strength and egg jelly on the potential for fertilization.
Supporting evidence from individual studies
A randomized, replicated study in 2005 of captive Fowler toads Bufo fowleri in the USA (Browne et al. 2006a) found that the proportion of eggs fertilized artificially was affected by hormone treatment used to stimulate egg production. Only treatments with lutenizing hormone-releasing hormone-a (LHRHa; 20 μg or more) plus another hormone resulted in fertilized eggs. The proportion of fertilized eggs was significantly higher following treatment with progesterone (5 mg) and 60 μg LHRHa (73%) than progesterone with 20 μg LHRHa and dopamine-2 receptor antagonist pimozide (35%) or progesterone with 60 μg LHRHa, pimozide and human chorionic gonadotrophin (500 IU; 20%). Following treatment with LHRHa but no progesterone only one toad produced eggs, of which 34% became fertilized. Second doses of 60 μg LHRHa or 500 IU human chorionic gonadotrophin given 24 or 48 hours after initial doses resulted in low egg numbers and fertilization. Wild-caught toads were housed in 50 x 40 x 10 cm tanks. Females were randomly assigned to treatments with seven females/treatment. Treatments were given in 100 μl of saline. Eggs were fertilized in a dish with spermic urine.
A replicated study in 2005 of captive Wyoming toad Bufo baxteri in the USA (Browne et al. 2006b) found that the proportion of eggs that became fertilized artificially was similar following one or two priming dose of hormones, but two priming doses resulted in higher numbers of viable eggs. Females given two priming doses produced significantly more tadpoles than those given one priming dose (2,300 vs 84). Toads were housed in 45 l tanks. Ten females were primed with 500 IU human chorionic gonadotrophin and 4 µg lutenizing hormone-releasing hormone (LHRHa). After 72 hours, the 10 females and an additional 10 females were given 100 IU human chorionic gonadotrophin and 0.8 µg LHRHa, followed 96 hours later by 500 IU human chorionic gonadotrophin and 4 µg LHRHa. Eggs produced during the fertile period (12–18 hours after hormone treatment) were fertilized in a dish with spermic urine.
A replicated study in 2009 of southern corroboree frogs Pseudophryne corroboree at Monash University, Australia (Byrne & Silla 2010) found that artificial fertilization resulted in 55% of eggs being fertilized, but embryos failed prior to gastrulation. Fertilization and the stage that the embryo failed varied between and within females. Hormone treatment was used to induce sperm and egg release. Artificial fertilization was attempted by combining spermic urine (1.1–2.9 x 102) with eggs from five females in a dilute solution of simplified amphibian Ringer solution at 10°C. Embryonic development was checked every 6–12 hours for seven days.
A randomized, replicated study in 2009 of captive Gϋnther’s toadlets Pseudophryne guentheri in Western Australia (Silla 2011) found that hormone treatment with one priming injection resulted in high artificial fertilization rates (91–100%), whereas eggs with zero or two priming treatments failed to fertilize. Twenty-four females were randomly assigned to three treatments: a single dose of 2 μ/g lutenizing hormone-releasing hormone-a in simplified amphibian Ringer solution, or a dose preceded by one or two priming injections of 0.4 μ/g lutenizing hormone-releasing hormone (one hour apart). Twenty eggs/female were fertilized with sperm from macerated testis of wild caught males in simplified amphibian Ringer solution.
- Browne R.K., Seratt J., Li H. & Kouba A. (2006) Progesterone improves the number and quality of hormonally induced fowler toad (Bufo fowleri) oocytes. Reproductive Biology and Endocrinology, 4, 1-7
- Browne R.K., Seratt J., Vance C. & Kouba A. (2006) Hormonal priming, induction of ovulation and in-vitro fertilization of the endangered Wyoming toad (Bufo baxteri). Reproductive Biology Endocrinology, 4, 34
- Byrne P.G. & Silla A.J. (2010) Hormonal induction of gamete release and in-vitro fertilisation in the critically endangered Southern Corroboree Frog, Pseudophryne corroboree. Reproductive Biology and Endocrinology, 8, 144
- Silla A.J. (2011) Effect of priming injections of luteinizing hormone-releasing hormone on spermiation and ovulation in Günther’s toadlet, Pseudophryne guentheri. Reproductive Biology and Endocrinology, 9, 68-76