Applying cryopreservation to anuran conservation biology

Poo, Sinlan; Hinkson, Kristin M.

doi:10.1111/csp2.91

Cited by 19 publications

(19 citation statements)

References 43 publications

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“…Frozen sperm can then be used in reproductive efforts to introduce old founder lines, new genetic variation, and build population numbers (Browne & Figiel Jr., 2011;Howell et al, 2020;Kouba et al, 2013). Thus far, cryopreservation has been used mainly in laboratory settings to store gametes of captive individuals held in assurance colonies (Browne et al, 1998;Poo & Hinkson, 2019;Shishova et al, 2011). However, cryopreservation protocols are now being developed to collect and freeze sperm from wild amphibians and incorporate the sperm through IVF into the captive collection for increased genetic variation instead of removing reproductively viable individuals.…”

Section: Introductionmentioning

confidence: 99%

Linking in‐situ and ex‐situ populations of threatened amphibians through genome banking

Burger

Julien

Kouba

et al. 2021

Conservat Sci and Prac

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Genetic variation of the endangered Puerto Rican crested toad (PRCT;Peltophryne lemur) has dwindled over time in both wild and captive populations, leading to long-term sustainability issues for the recovery program. To address this challenge, we propose that PRCTs can be used as a model species to show how in-situ and ex-situ populations can be linked through sperm biobanking and gamete transfer, expanding genetic variability of both populations. Male toads (n = 10) in Guayanilla, Puerto Rico were administered human chorionic-gonadotropin (hCG) and luteinizing-hormonereleasing-hormone analog (GnRHa) to stimulate spermiation. Sperm was collected noninvasively from 9/10 males, cryopreserved in 10% trehalose with either 10% N,N-dimethylformamide (DMFA) or 10% dimethyl sulfoxide (DMSO), and transported in liquid nitrogen vapor to the National Amphibian Genome Resource Bank at Mississippi State University. Ultrasonography was used to identify females (n = 3) with mature oocytes for GnRHa-induced oviposition of eggs for in-vitro fertilization (IVF). Post-thaw sperm motility was 28% and 25% for sperm cryopreserved with DMFA or DMSO, respectively. Of the 9,672 eggs used for IVF, 4% (n = 306/6,981) were fertilized with frozenthawed sperm, compared with 20% (n = 525/2691) fertilized with fresh sperm controls. Overall, 46 toadlets were produced from frozen-thawed sperm. After 4.5 months of headstarting, 14 juvenile toads produced from various genetic crosses using frozen-thawed sperm were released to new sites in the wild, introducing unique genetic representation and new founder lines. After 1.5 years, 24 adult toads produced using frozen-thawed sperm continue to thrive in the captive collection, and one of these males has now produced an F2 generation of offspring with 5,085 tadpoles released to the wild. This transformational study is the first to produce reproductively viable adult PRCTs using cryopreserved sperm from hormonally-induced wild males and captive females, introducing innovative methods that link in-situ and ex-situ populations of endangered amphibians to revolutionize genetic management.

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Section: Introductionmentioning

confidence: 99%

Linking in‐situ and ex‐situ populations of threatened amphibians through genome banking

Burger

Julien

Kouba

et al. 2021

Conservat Sci and Prac

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“…Sperm viability is an important indicator of fertilization capacity, obtaining sperm samples of maximum viability is therefore an important target for ensuring successful artificial fertilizations. Additionally, it is important that sperm suspensions are of high initial viability prior to cryopreservation, as current freeze-thaw protocols often result in a substantial loss of viability ( Poo and Hinkson, 2019 ). Sperm viability was high in response to the two optimum GnRH-a dose treatments: 1 μg injected or 100 μg GnRH-a topically applied, averaging 70 and 72% live sperm, respectively.…”

Section: Discussionmentioning

confidence: 99%

The effect of injection and topical application of hCG and GnRH agonist to induce sperm-release in the roseate frog, Geocrinia rosea

Silla

Roberts

Byrne

2020

Conservation Physiology

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Reproductive technologies may assist amphibian conservation breeding programs (CBPs) to achieve propagation targets and genetic management goals. However, a trial-and-error approach to protocol refinement has led to few amphibian CBPs routinely employing reproductive technologies with predictable outcomes. Additionally, while injections can be safely administered to amphibians, perceived animal welfare risks, such as injury and disease transmission, warrant the development of alternative hormone administration protocols. The present study investigated the spermiation response of roseate frogs, Geocrinia rosea, administered various doses of human chorionic gonadotropin (hCG) and gonadotropin-releasing hormone agonist (GnRH-a) via subcutaneous injection. This study also quantified the spermiation response of frogs administered both hormones via topical application. Total sperm, sperm concentration and sperm viability were assessed over a 12-h period post hormone administration. Males released sperm in response to the injection of hCG (88–100% response; 5, 10 or 20 IU), but all samples collected from males administered hCG topically (100, 100 + DMSO or 200 IU hCG) were aspermic. In contrast, males consistently released sperm in response to both the injection (100% response; 1, 5 or 10 μg), or topical application (80–100% response; 50, 50 + DMSO or 100 μg) of GnRH-a. Overall, the administration of GnRH-a was more effective at inducing spermiation than hCG. Mean total sperm and sperm concentration were highest in response to the optimal topically applied dose of 100 μg GnRH-a (mean total sperm = 2.44 × 103, sperm concentration = 1.48 × 105 sperm/ml). We provide novel evidence that topical application provides a viable alternative to injection for the administration of GnRH-a to induce spermiation in amphibians.

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“…Hormone dosages were based on Kouba et al . (2012) and Poo & Hinkson (2019). Sperm were collected through the urine using a 1‐mm‐diameter catheter one hour after hormone injections.…”

Section: Methodsmentioning

confidence: 99%

“…Because efforts to facilitate natural breeding by dusky gopher frogs in captivity have not been successful, assisted reproductive technologies were used to breed adults in the captive population at the Memphis Zoo to produce juvenile frogs for this study. This involved administering hormones to induce ovulation and spermiation, and fertilizing the eggs in vitro (Kouba et al ., 2012; Poo & Hinkson, 2019). To induce oviposition, two priming doses of 3.7 IU hCG/g body weight were administered 72 h apart (hCG: human chorionic gonadotropin; Sigma‐Aldrich, St. Louis, MO), followed by an ovulation dose of 13.5 IU Hcg + 0.4µg GnRH/g body mass 24 h after the second priming dose (GnRH: gonadotropin‐releasing hormone; Sigma‐Aldrich, St. Louis, MO).…”

Section: Methodsmentioning

confidence: 99%

Survival, movements and habitat use of captive‐bred and reintroduced dusky gopher frogs

Roznik

Reichling

2020

Animal Conservation

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Despite the growing roles of assisted reproduction and captive breeding in reintroductions, we have a limited understanding of the viability of these animals once released. We radiotracked endangered dusky gopher frogs Lithobates sevosus for up to 24 days after they were released into the wild to (1) assess the viability of juvenile frogs produced using artificial fertilization and raised in captivity until four weeks post‐metamorphosis, (2) investigate survival rates, movement patterns, and habitat use and (3) examine whether it is more beneficial to release frogs at the pond edge or into burrows in the uplands. Released frogs had a relatively high survival rate (76% overall, with mortality ceasing after 14 days) and behaved as expected, moving fairly long distances (up to 153 m daily and 426 m total) and locating suitable shelters, including burrows and stump holes. Frogs that sheltered underground had a 22% higher survival rate than frogs that did not, highlighting the importance of these habitat features. Compared to frogs released into burrows, frogs released at the pond had a 33% lower survival rate, spent less time underground and moved farther and more often, likely while searching for shelter. Frogs selected habitat characteristics that are associated with effects of fire, including an open canopy and abundant ground vegetation. Our study demonstrates that juvenile dusky gopher frogs successfully transitioned to their natural habitat, and that any effects of artificial fertilization or captivity on juvenile viability appear to be minimal. Releasing frogs into areas with abundant underground refuges and using frequent prescribed fire to maintain the habitat should benefit dusky gopher frog populations and improve the success of reintroductions.

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Applying cryopreservation to anuran conservation biology

Cited by 19 publications

References 43 publications

Linking in‐situ and ex‐situ populations of threatened amphibians through genome banking

Linking in‐situ and ex‐situ populations of threatened amphibians through genome banking

The effect of injection and topical application of hCG and GnRH agonist to induce sperm-release in the roseate frog, Geocrinia rosea

Survival, movements and habitat use of captive‐bred and reintroduced dusky gopher frogs

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