Action: Use hormone treatment to induce sperm and egg release during captive breeding
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- One review and nine of 10 replicated studies (including two randomized, controlled studies) in Austria, Australia, China, Latvia, Russia and the USA found that hormone treatment of male amphibians stimulated or increased sperm production (Mansour, Lahnsteiner & Patzner 2010, Silla 2011) or resulted in successful breeding in captivity. One found that hormone treatment of males and females did not result in breeding. Four found that the amount and viability of sperm produced was affected by the type, amount or number of doses of hormone.
- One review and nine of 14 replicated studies (including six randomized and/or controlled studies) in Australia, Canada, China, Ecuador, Latvia and the USA found that hormone treatment of female amphibians had mixed results, with 30–71% of females producing viable eggs following treatment, or with egg production depending on the combination, amount or number of doses of hormones. Three found that hormone treatment stimulated egg production or successful breeding in captivity. Two found that hormone treatment did not stimulate or increase egg production.
- Five replicated studies (including one controlled study) in Canada, Latvia and the USA found that eggs induced by hormone treatment were raised successfully to tadpoles, toadlets or froglets in captivity. Two replicated studies, one of which was small, in Ecuador and the USA found that most toads died before or soon after hatching.
Captive animals do not always breed successfully under artificial conditions. Reproductive technologies such as hormone treatment to induce ovulation or sperm production are techniques that can be used in an attempt to achieve or increase breeding success by amphibians in captive facilities. Hormone stimulation protocols are often species specific.
Supporting evidence from individual studies
A replicated study in 1983 of Puerto Rican toads Peltophryne lemur at Buffalo Zoological Gardens, USA (Miller 1985) found that following hormone treatment, one of three females produced viable eggs. Another female produced hundreds of infertile eggs and the third a few unfertilized eggs. Over 150 tadpoles hatched from the viable clutch and tadpole survival was 100%. In 1983, 75 of the toadlets were released at an artificial pond in Puerto Rico. Three male and female captive bred-toads were housed in an enclosure. Breeding was induced by lutenizing hormone-releasing hormone (0.01–0.05 ml/10 g body weight). Tadpoles were transferred to aquaria for rearing and metamorphs to containers.
A small, replicated study in 1983 of Puerto Rican crested toads Bufo lemur in the USA (Paine 1985) found that hormone treatment of males and females did not induce successful breeding in captivity. Two pairs displayed breeding behaviour for three weeks but no eggs were produced. Adults were housed in wooden cages (85 x 90 x 75 cm) with bark chips, wood, plants and a pool. To induce breeding, humid conditions were created by placing toads in glass tanks (76 x 70 x 200 cm) with an overnight misting system. Lutenizing hormone-releasing hormone was given under the skin to females (0.1 ml/100 g body weight) and males (0.01ml/100 g).
A review of captive breeding programmes (Maruska 1986) found that breeding was induced with gonadotrophin-releasing hormone in White’s tree frog Litoria caerulea and African red frog Phrynomerus bifasciatus.
A replicated study in 1991–1994 of Wyoming toads Bufo hemiophrys baxteri in a zoo in Colorado, USA (Burton et al. 1995) found that five of seven hormonally induced females produced thousands of fertile eggs in 1994. However, the majority of tadpoles that hatched died within 72 hours. Deaths were considered by the authors to have been due to water quality. In captivity, two to four wild-caught and captive-bred toads were housed per tank (40 x 61 x 23 cm) at 20°C. Cork bark, sheet moss, sand, water, artificial plants and a basking lamp were provided. In 1991–1993, toads were transported to breeding enclosures at the edge of the lake. In 1994, five toads were overwintered for six weeks at 4.5°C. Seven females were hormonally induced and paired in captivity.
A replicated study in 1988–1992 of captive European tree frogs Hyla arborea in Latvia (Zvirgzds, Stašuls & Vilnìtis 1995) found that following hormone treatment of males and females, frogs bred successfully in captivity. In several cases three or four hormone injections were required to induce spawning. Females each produced 200–800 eggs. An average of 60–90% of larvae metamorphosed in captivity. The period of metamorphosis was shorter in captivity than the wild (30–60 vs 90 days). Over 4,000 froglets were produced. Wild-caught frogs were housed in outdoor tanks and were overwintered in a refrigerator. From February, daylight period, UV light and feeding was increased. Two males and one female were placed in separate 35 l aquaria with water and plants. Breeding was stimulated with hormone injections (100 mg luliberin-surphagon/ml of solution) in March or May. Females received 15–20 mg and males 10 mg. The injection was repeated after 24 hours if spawning did not start. Larvae, metamorphs and toadlets were raised in separate tanks.
A small, replicated, controlled study of captive Chinese giant salamanders Andrias davidianus in China (Xiao et al. 2006) found that injection with reproductive hormones induced egg and sperm production. Eggs were produced by 60% of females given injections of lutenizing hormone-releasing hormone-a or human chorionic gonadotrophin (400–500 eggs). However, mating was not observed. Eggs were laid earlier following injection with human chorionic gonadotrophin compared to lutenizing hormone-releasing hormone and earlier with higher water temperatures. A single injection was more effective than repeated injections. Females produced eggs between 96–120 hours and sperm was produced after 80 hours. A 1°C drop in water temperature resulted in a 10 hour delay. Animals not injected with hormones did not produce eggs or sperm and reproductive organs degenerated and were absorbed. Wild-caught salamanders were housed in 16 m2 tanks.
A randomized, replicated study in 2005 of captive Fowler toads Bufo fowleri in the USA (Browne et al. 2006a) found that treatments of progesterone along with other hormones were effective at inducing egg production in a high proportion of toads and resulted in high egg numbers. Successful progesterone (5 mg) treatments were: progesterone and lutenizing hormone-releasing hormone-a (LHRHa; 60 μg) alone (71% produced eggs; 2,004 eggs/toad), or with dopamine-2 receptor antagonist pimozide (0.25 mg) and human chorionic gonadotrophin (500 IU; 85%; 1,078), or progesterone, LHRHa (20 μg) and pimozide (0.25 mg; 58%; 2,486). Two repeated doses of 5 mg progesterone or a single dose of 20 μg LHRHa did not result in egg production. Egg production was low with 4 μg LHRHa and 500 IU human chorionic gonadotrophin (29%; 2,283) or 20 μg LHRHa and 0.25 mg pimozide (14%; 627). 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. Wild caught toads were housed in 50 x 40 x 10 cm tanks. Females were randomly assigned to the seven treatments with seven females/treatment. Treatments were given in 100 μl of saline.
A replicated study in 2005 of captive Wyoming toad Bufo baxteri in the USA (Browne et al. 2006b) found that one or two priming doses of hormones were required to induce egg production, but not sperm production. Eight of 10 males receiving a single dose of 300 IU human chorionic gonadotrophin (hCG) produced spermic urine within five hours. Females given a single dose of hCG plus lutenizing hormone-releasing hormone-a (LHRHa) produced no eggs. Compared to one priming dose, two priming doses resulted in a greater proportion of females spawning (70 vs 88%) and significantly higher average number of eggs produced (1,647 vs 3,280) and numbers produced/female at a given time (4 vs 7). The total number of eggs/female did not differ with treatment. Toads were housed in 45 l tanks. Ten females were primed with 500 IU hCG and 4 µg LHRHa. After 72 hours, the 10 females and an additional 10 females were given 100 IU hCG and 0.8 µg LHRHa, followed 96 hours later by 500 IU hCG and 4 µg LHRHa.
A replicated study in 1999–2006 of Wyoming toads Bufo hemiophrys baxteri in Saratoga, Wyoming, USA (Springer 2007) found that hormone treatment of males and females induced successful breeding in captivity. Between 1999 and 2006, an average of 6,863 toads were bred and released each year. In 2006, an 18% increase in hatch rate was achieved. This was thought to be due to over-wintering at cooler temperatures, to simulate the harsh weather faced in the wild. Breeding pairs were carefully selected from a studbook of the 150 captive toads. Pairs were housed in separate water tanks. Toads were injected with hormones to induce production of eggs and sperm. Over 20 breeding events were undertaken each year. Most toads are released as tadpoles in autumn.
A randomized, replicated, controlled study in 2009 of southern corroboree frogs Pseudophryne corroboree at Monash University, Australia (Byrne & Silla 2010) found that hormone treatment successfully induced sperm release and to a lesser extent egg production. Human chorionic gonadotropin (hCG) and luteinizing hormone-releasing hormone (LHRHa) both induced significantly higher proportions of males to release sperm than controls (82 vs 0%). LHRHa treated males released significantly higher numbers of sperm (670 vs 50) and concentration of sperm (4,500 vs 800 x 103/ml) over a longer period than those treated with human chorionic gonadotropin. There was no significant difference in numbers of females releasing eggs following LHRHa and controls (30 vs 0%). Eggs were released 24–48 hours post-treatment (peak 36 hours). Average clutch size was 15. Six randomly selected males were given a dose of either 20 μg/g bodyweight of human chorionic gonadotropin or 5 μg/g of LHRHa in simplified amphibian Ringer solution (SAR) or a control of 0.1 ml of SAR. Sperm response was tested in urine seven times up to 72 hours post-treatment. Seventeen females received a priming (1 μg/g) and ovulatory dose (5 μg/g) of LHRHa in SAR. Eight received a control of 0.1 ml of SAR. Ovulation was tested every 12 hours for five days.
A replicated study in 2009 of captive European common frogs Rana temporaria in Austria (Mansour, Lahnsteiner & Patzner 2010) found that injecting males with human chorionic gonadotrophin increased sperm production. Males stimulated with hormones had greater sperm production than untreated males (0.004 vs 0.002 testis/body weight). The same was true for the sperm cell concentration (80 vs 11 x 106/ml in 1.5 ml motility-inhibiting saline/testes). Males received injections of 150 IU of human chorionic gonadotrophin and were killed after 15 hours. Testes were removed weighed and macerated in motility-inhibiting saline.
A replicated, controlled study in 2009 of captive Gϋnther’s toadlets Pseudophryne guentheri in Western Australia (Silla 2010) found that hormone treatment successfully induced sperm release. Luteinizing hormone-releasing hormone (LHRHa) in doses of 1, 2, 4 or 8 μg/g induced 100% of males to produce sperm, compared to 10–30% of controls. Numbers of sperm released was significantly higher following 2 μg/g of LHRHa (25 x 103) than 8 μg/g (5 x 103) or controls (0); other doses did not differ significantly (8–12 x 103). Sperm viability was significantly higher following the 1 μg/g compared to 8 μg/g treatment. Arg8-vasotocin acetate salt (4 μg/g) alone or with 2 μg/g LHRHa resulted in similar numbers of males releasing sperm as a single 2 μg/g dose of LHRHa (71; 71; 100% respectively). However, sperm numbers were significantly lower (0 vs 25 x 103). Male toadlets were given a single dose of 1, 2, 4 or 8 μg/g bodyweight of LHRHa in simplified amphibian Ringer solution, or a control of 100 μL of simplified amphibian Ringer solution (n = 7–10/treatment). Sperm release was tested at 3, 7 and 12 hours post-treatment.
A replicated, controlled study in 2008 of captive frogs in Ottawa, USA (Trudeau et al. 2010) found that injection with a gonadotropin-releasing hormone (GnRH) agonist and a dopamine antagonist was effective at inducing egg production. After one week in captivity GnRH-A (0.4 μg/g body weight) and metoclopramide (10 μg/g) was more effective at inducing egg production in northern leopard frogs Lithobates pipiens (100%) than GnRH-A and pimozide (10 μg/g; 50%), GnRH-B (0.4 μg/g) and pimozide (42%) or no treatment (0%). After one month in captivity, GnRH-A with 10 μg/g of metoclopramide was significantly more effective than with 5 μg/g (60 vs 44%). Out-of-season breeding was induced with GnRH-A and metoclopramide in five pairs, with 25% of females producing eggs (and metamorphs). Egg production (and metamorphs) was also induced in Argentine horned frog Ceratophrys ornate (1 pair), Cranwell's horned frog Ceratophrys cranwelli (1 pair) and escuercitos Odontophrynus americanus (10 males, 5 females). A week after collection in April, 12 female and 18 male leopard frogs were given one of four initial treatments. Controls were given saline and dimethyl sulfoxide. A month after collection, nine females and 15 males were given 0.4 μg/g GnRH-A and either 5 or 10 μg/g of metoclopramide, or were controls. Following collection in September, artificial overwintering was induced in eight females and 15 males. In October, males were primed with two injections of GnRH-A (0.025 then 0.05 μg/g a week later). Frogs were then injected with GnRH-A (0.4 μg/g) and metoclopramide (10 μg/g).
A randomized, replicated, controlled study in 2009 of captive Gϋnther’s toadlets Pseudophryne guentheri in Western Australia (Silla 2011) found that hormone treatment successfully induced sperm and egg release. Proportions of males producing sperm with no, one or two priming injections of luteinizing hormone-releasing hormone (LHRHa) did not differ (100%), but were significantly higher than controls (25%). Amount of sperm produced decreased with priming treatments (none: 1.8 x 104; one: 0.6 x 104; two: 0.3 x 104). Sperm viability did not differ between hormone treatments (0.6–0.7 sperm/total) and was highest at 3 hours. Significantly higher numbers released eggs with one or two priming treatments (priming: 100%; none: 25%; control: 0%). The same was true for the number of eggs (priming: 217–220; none: 19; control: 0). Mass of eggs from two priming treatments was significantly greater than from no priming (0.007 vs 0.001 g; one priming: 0.006 g). Thirty-two wild collected males and females were randomly assigned to four treatments: a single dose of 2 μ/g LHRHa in simplified amphibian Ringer solution, or a dose preceded by one or two priming injections of 0.4 μ/g LHRHa (hour apart), or a control of 100 μ/g of simplified amphibian Ringer solution. Sperm release was tested at 3, 7 and 12 hours after treatment. Ovulation was tested at 10–11 hours.
A replicated study in 2010–2011 of captive Oregon spotted frogs Rana pretiosa in Vancouver, Canada (Thoney 2011) found that frogs bred successfully in captivity and that treatment with hormones did not increase the proportion of females producing eggs or numbers of eggs. The two hormonal substances tested did synchronize timing of egg production. The small number of mature frogs produced 291 tadpoles in the first year. In 2011, a larger number of frogs bred and over 9,000 eggs were produced, of which 3,000 hatched. Providing a seasonal daylight and temperature regime was considered by the authors to be crucial to breeding success. Metamorphs and tadpoles were released in spring 2011. Eggs were collected each year from the wild to increase genetic diversity of the captive population.
A small, replicated, controlled study in 2011–2012 of captive harlequin toads Atelopus in Ecuador (Coloma & Almeida-Reinoso 2012) found that following treatment with human chorionic gonadotrophin, females produced eggs. The one female elegant stubfoot toad Atelopus elegans and one Pebas stubfoot toad Atelopus spumarius treated with hormones produced a clutch of eggs. However, most Pebas stubfoot toad embryos were dead within eight days. Two untreated elegant stubfoot toads did not produce eggs. Twenty adult elegant stubfoot toads and eight Pebas stubfoot toads were wild caught. Breeding tanks were 60 x 35 x 30 cm with stones, plants and an open system of filtered water. One of three female elegant stubfoot toads and one Pebas stubfoot toad were stimulated with human chorionic gonadotrophin (0.05 ml).
A replicated, controlled study in 2011 of captive amphibians in Russia (Uteshev et al. 2012) found that the greatest sperm production was induced with high dose lutenizing hormone-releasing hormone-a (LHRHa) for common frogs Rana temporaria and priming with LHRHa prior to human chorionic gonadotrophin (hCG) for common toads Bufo bufo. In common frogs, 1.2 μg/g bodyweight LHRHa induced significantly higher sperm numbers (650 x 106/ml) than pituitary extract (485 x 106) or 0.12 μg/g LHRHa (444 x 106), which produced significantly higher numbers than 23 IU/g hCG (170 x 106) and 12 IU/g hCG (39 x 106). High dose LHRHa had the highest percentage of samples with sperm concentrations above 200 x 106/ml (high LHRH: 40%; pituitaries: 36%; low LHRH: 15%; hCG: 0%). Sperm motility was similar with all treatments (76–90%). Priming common toads resulted in significantly higher numbers (11.6 x 106 vs 8.0 x 106/ml) and quality of sperm (motility: 85 vs 73%), but not higher sperm concentration (1.5 x 106 vs 1.8 x 106/ml). Four wild-caught frogs received each of the five hormone injection treatments. There were also 10 controls. Four wild-caught toads were primed with 0.13 μg/g LHRHa 24 hours before receiving 13 IU/g hCG; controls received only the second dose. Spermic urine was monitored.
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- Coloma L.A. & Almeida-Reinoso D. (2012) Ex situ management of five extant species of Atelopus in Ecuador - progress report. Amphibian Ark Newsletter, 20, 9-12
- Uteshev V.K., Shishova N.V., Kaurova S.A., Browne R.K. & Gakhova E.N. (2012) Hormonal induction of spermatozoa from amphibians with Rana temporaria and Bufo bufo as anuran models. Reproduction, Fertility and Development, 24, 599–607