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Animals
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Reproductive Biology and Assisted Reproductive Technologies in Amphibians
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Reproductive Biology and Assisted Reproductive Technologies in Amphibians

A special issue of Animals (ISSN 2076-2615). This special issue belongs to the section "Herpetology".

Deadline for manuscript submissions: closed (31 July 2023) | Viewed by 11319

Special Issue Editor

Dr. Lucía Arregui

E-Mail Website
Guest Editor
Aquatic Germplasm and Genetic Resources Center, School of Renewable Natural Resources, Louisiana State University Agricultural Center, Baton Rouge, LA 70820, USA
Interests: reproductive biology; sperm quality; spermatogenesis; assisted reproductive technologies

Special Issue Information

Dear Colleagues,

Conservation of amphibians is one of the greatest challenges of the century. Amphibians are facing a dramatic worldwide extinction crisis on a scale not previously seen due to anthropogenic impacts. The integration of multiple disciplines for the development and implementation of assisted reproductive technologies and for enhancing the knowledge and understanding on basic reproductive biology for a greater number of amphibians species is crucial to boost global conservation efforts.

Assisted reproductive technologies could enhance reproductive success, reduce the number of live animals kept in captivity, facilitate selective breeding (that allow maintaining and promoting genetic diversity and linking ex situ and in situ populations), and enable long-term storage of reproductive material. In addition, understanding the natural reproductive behavior of a species and how it responds to changes in the environment is crucial for the application of these technologies and the success of in situ and ex situ conservation approaches.

The aim of this Special Issue is to publish original research articles and reviews which cover any aspect of reproduction in amphibians that contribute to the practical grand challenges of conservation. Manuscripts concerning amphibian reproductive behavior, physiology and endocrinology, assisted reproductive techniques, and germplasm storage are invited for this Special Issue.

Dr. Lucía Arregui
Guest Editor

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Animals is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2400 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • cryopreservation
  • in vitro fertilization
  • reproductive behavior
  • reproductive mode
  • reproductive hormones

Published Papers (7 papers)

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Research

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18 pages, 4642 KiB  
Article
Establishment of a Practical Sperm Cryopreservation Pathway for the Axolotl (Ambystoma mexicanum): A Community-Level Approach to Germplasm Repository Development
by Nicholas Coxe, Yue Liu, Lucía Arregui, Rose Upton, Sarah Bodenstein, Steven Randal Voss, Maria T. Gutierrez-Wing and Terrence R. Tiersch
Animals 2024, 14(2), 206; https://doi.org/10.3390/ani14020206 - 08 Jan 2024
Viewed by 940
Abstract
The axolotl (Ambystoma mexicanum) draws great attention around the world for its importance as a biomedical research model, but housing and maintaining live animals is increasingly expensive and risky as new transgenic lines are developed. The goal of this work was [...] Read more.
The axolotl (Ambystoma mexicanum) draws great attention around the world for its importance as a biomedical research model, but housing and maintaining live animals is increasingly expensive and risky as new transgenic lines are developed. The goal of this work was to develop an initial practical pathway for sperm cryopreservation to support germplasm repository development. The present study assembled a pathway through the investigation of axolotl sperm collection by stripping, refrigerated storage in various osmotic pressures, cryopreservation in various cryoprotectants, and in vitro fertilization using thawed sperm. By the stripping of males, 25–800 µL of sperm fluid was collected at concentrations of 1.6 × 106 to 8.9 × 107 sperm/mL. Sperm remained motile for 5 d in Hanks’ Balanced Salt Solution (HBSS) at osmolalities of 100–600 mOsm/kg. Sperm cryopreserved in 0.25 mL French straws at 20 °C/min in a final concentration of 5% DMFA plus 200 mM trehalose and thawed at 25 °C for 15 s resulted in 52 ± 12% total post-thaw motility. In six in vitro fertilization trials, 20% of eggs tested with thawed sperm continued to develop to stage 7–8 after 24 h, and a third of those embryos (58) hatched. This work is the first report of successful production of axolotl offspring with cryopreserved sperm, providing a general framework for pathway development to establish Ambystoma germplasm repositories for future research and applications. Full article
(This article belongs to the Special Issue Reproductive Biology and Assisted Reproductive Technologies in Amphibians)
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17 pages, 1683 KiB  
Article
Cryopreservation Cooling Rate Impacts Post-Thaw Sperm Motility and Survival in Litoria booroolongensis
by Rebecca J. Hobbs, Rose Upton, Natalie E. Calatayud, Aimee J. Silla, Jonathan Daly, Michael S. McFadden and Justine K. O’Brien
Animals 2023, 13(19), 3014; https://doi.org/10.3390/ani13193014 - 25 Sep 2023
Cited by 1 | Viewed by 930
Abstract
The cryopreservation and storage of gametes (biobanking) can provide a long-term, low-cost option for the preservation of population genetic diversity and is particularly impactful when applied to manage selective breeding within conservation breeding programs (CBPs). This study aimed to develop a sperm cryopreservation [...] Read more.
The cryopreservation and storage of gametes (biobanking) can provide a long-term, low-cost option for the preservation of population genetic diversity and is particularly impactful when applied to manage selective breeding within conservation breeding programs (CBPs). This study aimed to develop a sperm cryopreservation protocol for the critically endangered Booroolong frog (Litoria booroolongensis) to capture founder genetics within the recently established (est. 2019) CBP for this species. Hormone-induced sperm release was achieved using established protocols, and spermic urine samples were collected over a 6-h period. Pooled spermic urine samples (n = 3 males) were divided equally between two cryoprotectant (CPA) treatments and diluted by 1:5 (sperm:CPA) with either 15% (v/v) dimethyl sulfoxide + 1% (w/v) sucrose in simplified amphibian Ringer’s (SAR; CPAA) or 10% (v/v) dimethylformamide + 10% (w/v) trehalose dihydrate in SAR (CPAB). The samples were cryopreserved in 0.25 mL straws using either a programmable freezer (FrA) or an adapted dry shipper method (FrB). The thawed samples were activated via dilution in water and assessed for viability and motility using both manual assessment and computer-assisted sperm analysis (CASA; 0 h, 0.5 h post-thaw). Upon activation, the survival and recovery of motility (total motility, forward progression and velocity) of cryopreserved sperm suspensions were higher for sperm preserved using FrB than FrA, regardless of CPA composition. This work supports our long-term goal to pioneer the integration of biobanked cryopreserved sperm with population genetic management to maximize restoration program outcomes for Australian amphibian species. Full article
(This article belongs to the Special Issue Reproductive Biology and Assisted Reproductive Technologies in Amphibians)
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12 pages, 718 KiB  
Article
Gamete Collection, Artificial Fertilization and Captive-Rearing of Eggs in a Terrestrial-Breeding Anuran with Parental Care: Alytes obstetricans
by Lucía Arregui and Jaime Bosch
Animals 2023, 13(17), 2802; https://doi.org/10.3390/ani13172802 - 04 Sep 2023
Viewed by 1065
Abstract
The genus Alytes consists of six primitive terrestrial-breeding species (five of them endangered or present in restricted distributions) with unique male parental care. Alytes obstetricans was used as a model for the development of assisted reproductive technologies for gamete collection, artificial fertilization and [...] Read more.
The genus Alytes consists of six primitive terrestrial-breeding species (five of them endangered or present in restricted distributions) with unique male parental care. Alytes obstetricans was used as a model for the development of assisted reproductive technologies for gamete collection, artificial fertilization and captive-rearing of embryos. Sperm collection was achieved with human chorionic gonadotropin (hCG), but only 17 to 50% of males responded to the hormone. In addition, an effect of captivity on sperm quality was observed. A combination of gonadotropin-releasing hormone agonist (GnRHa) and hCG was tested for induction of ovulation, and 100% of females responded to the treatment. Recently collected sperm was used in artificial fertilization, and 10% of eggs showed complete embryo development. To design a method for captive-rearing of embryos, natural clutches (n = 13) were removed from males and divided into two sub-clutches. One was kept with all eggs together and in the other, the egg string was cut and eggs were split individually. All sub-clutches with eggs kept separated presented embryo survival (ranging from 27 to 61%) that hatched normally, while only one sub-clutch with embryos maintained together had some embryo survival (36%). These results may contribute to the management of this and related species. Full article
(This article belongs to the Special Issue Reproductive Biology and Assisted Reproductive Technologies in Amphibians)
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22 pages, 1700 KiB  
Article
Recovery and Characterization of Spermatozoa in a Neotropical, Terrestrial, Direct-Developing Riparian Frog (Craugastor evanesco) through Hormonal Stimulation
by Yineska Otero, Natalie E. Calatayud, Igli D. Arcia, Denise Mariscal, Diego Samaniego, Dionel Rodríguez, Karina Rodríguez, Jorge Guerrel, Roberto Ibáñez and Gina Della Togna
Animals 2023, 13(17), 2689; https://doi.org/10.3390/ani13172689 - 22 Aug 2023
Cited by 1 | Viewed by 915
Abstract
The Vanishing Rainfrog (Craugastor evanesco) is an endemic and critically endangered frog species of Panama. It is suspected that 90% of the population has disappeared from the wild. Frogs were collected from the wild and brought to a Captive Breeding Program; [...] Read more.
The Vanishing Rainfrog (Craugastor evanesco) is an endemic and critically endangered frog species of Panama. It is suspected that 90% of the population has disappeared from the wild. Frogs were collected from the wild and brought to a Captive Breeding Program; however, accomplishing regular reproductive events for this species has been difficult. The objective of this study was to determine the effect of hormonal stimulation on the production and quality of C. evanesco spermatozoa, aiming to develop an efficient and safe sperm collection protocol as a tool to help reproduce this endangered species. Mature males received intra-peritoneal injections with one of six hormone treatments, including des-Gly10, D-Ala6, Pro-NHEt9—GnRH-A, Amphiplex or hCG. Urine samples were collected at 10 different time points post-injection. Quality assessments included sperm concentration, percentage motility, percentage forward progressive motility (FPM), osmolality, pH and morphology analysis. Our results indicate that the optimal treatment for the collection of highly concentrated sperm samples of C. evanesco is 4 µg/gbw GnRH, followed by Amphiplex and 2 µg/gbw GnRH as sub-optimal treatments and finally, 6 µg/gbw GnRH and 5 and 10 IU/gbw hCG as non-optimal treatments. GnRH-A at 4 μg/gbw and Amphiplex stimulated the production of samples with the highest sperm concentrations and quality, despite Amphiplex producing lower percentages of intact acrosome and tail. In contrast, hCG concentrations were not reliable inducers of sperm production, consistently showing lower concentrations, higher percentages of sperm abnormalities and more acidic spermic urine than that induced by Amphiplex and GnRH-A. Morphological assessments revealed that C. evanesco spermatozoa have a filiform shape with a large acrosome on the anterior part of an elongated head, a small midpiece and a long tail with two filaments joined together by an undulating membrane. Full article
(This article belongs to the Special Issue Reproductive Biology and Assisted Reproductive Technologies in Amphibians)
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11 pages, 1122 KiB  
Article
Application of Reproductive Technologies to the Critically Endangered Baw Baw Frog, Philoria frosti
by Aimee J. Silla, Rebecca J. Hobbs, Deon J. Gilbert, Damian Goodall, Marissa L. Parrott, Adam Lee, Justine K. O’Brien and Phillip G. Byrne
Animals 2023, 13(13), 2232; https://doi.org/10.3390/ani13132232 - 07 Jul 2023
Cited by 1 | Viewed by 1733
Abstract
Reproductive technologies (RTs) can assist integrated conservation breeding programs to attain propagation targets and manage genetic diversity more effectively. While the application of RTs to enhance the conservation management of threatened amphibians has lagged behind that of other taxonomic groups, a recent surge [...] Read more.
Reproductive technologies (RTs) can assist integrated conservation breeding programs to attain propagation targets and manage genetic diversity more effectively. While the application of RTs to enhance the conservation management of threatened amphibians has lagged behind that of other taxonomic groups, a recent surge in research is narrowing the divide. The present study reports on the first application of RTs (hormone-induced spawning, hormone-induced sperm-release, and sperm cryopreservation) to the critically endangered Baw Baw frog, Philoria frosti. To determine the effect of hormone therapy on spawning success, male–female pairs were administered either 0 μg/g gonadotropin-releasing hormone agonist (GnRHa), 0.5 μg/g GnRHa, or 0.5 μg/g GnRHa + 10 μg/g metoclopramide (MET) (n = 6–7 pairs/treatment), and the number of pairs ovipositing, total eggs, and percent fertilisation success were quantified. To determine the effect of hormone therapy on sperm-release and to establish the peak time to collect sperm post-hormone administration, males were administered 0 IU/g (n = 4), or 20 IU/g hCG (n = 16). Total sperm, sperm concentration, and percent viability were quantified at 0, 2, 4, 6, 8, 10, and 12 h post-hormone administration. Overall, the percentage of pairs ovipositing was highest in the GnRHa + MET treatment, with 71% of pairs ovipositing, compared to 57% and 33% of pairs in the GnRHa and control treatments, respectively. The quantity of sperm released from males in response to hCG peaked at 4 h post-hormone administration, though it remained high up to 12 h. The percent sperm viability also peaked at 4 h post-administration (94.5%), exhibiting a steady decline thereafter, though viability remained above 77% throughout the 12 h collection period. The remaining sperm samples (n = 22) were cryopreserved using established protocols and biobanked for long-term storage and future conservation applications. The mean post-thaw sperm viability was 59%, and the percent total motility was 17%. The results from this preliminary study will direct further applications of RTs to the critically endangered Baw Baw frog to assist with species recovery. Full article
(This article belongs to the Special Issue Reproductive Biology and Assisted Reproductive Technologies in Amphibians)
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8 pages, 564 KiB  
Communication
Effects of Water Temperature on Gonads Growth in Ambystoma mexicanum Axolotl Salamanders
by Chester R. Figiel, Jr.
Animals 2023, 13(5), 874; https://doi.org/10.3390/ani13050874 - 28 Feb 2023
Cited by 1 | Viewed by 2856
Abstract
The thermal environment is a major factor influencing amphibians. For example, amphibian reproduction occurs in specific temperatures, and minor changes in this aspect could have negative impacts on this biological process. Understanding the potential effects of temperature on reproductive output is important from [...] Read more.
The thermal environment is a major factor influencing amphibians. For example, amphibian reproduction occurs in specific temperatures, and minor changes in this aspect could have negative impacts on this biological process. Understanding the potential effects of temperature on reproductive output is important from both an ecological and captive breeding colony point of view. I examined temperature effects on reproduction in axolotl reared from egg to adult at 4 temperatures (15 °C, 19 °C, 23 °C, and 27 °C) These adult axolotls (n = 174) were measured and weighed, dissected, and their gonads were removed and weighed to obtain an individual’s reproductive allocation. Female axolotls reared at 23 °C had a greater Gonadosomatic Index (GSI) compared to axolotl reared at each of the other temperatures with axolotls reared at 27 °C having the lowest reproductive output. Moreover, all GSI pair-wise comparisons in the four temperature treatments were significantly different from each other (ANOVA, F (3, 66) = 61.681, p < 0.0001). Additionally male rearing temperature significantly influenced GSI (ANOVA, F (3, 89) = 10.441, p < 0.0001). Male axolotls reared at 19 °C had significantly greater GSI compared to males reared at the three other temperatures. There were no statistical differences among each of the other pair-wise comparisons. As seen in this experiment, axolotls may be especially sensitive to climate-driven warming due to their highly permeable skin and paedomorphic life history. Understanding how axolotls and other amphibian species adjust to the challenges of climate change is important in the management of this imperiled taxa. Full article
(This article belongs to the Special Issue Reproductive Biology and Assisted Reproductive Technologies in Amphibians)
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Review

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40 pages, 538 KiB  
Review
The Increasing Role of Short-Term Sperm Storage and Cryopreservation in Conserving Threatened Amphibian Species
by Zara M. Anastas, Phillip G. Byrne, Justine K. O’Brien, Rebecca J. Hobbs, Rose Upton and Aimee J. Silla
Animals 2023, 13(13), 2094; https://doi.org/10.3390/ani13132094 - 24 Jun 2023
Cited by 6 | Viewed by 1670
Abstract
Multidisciplinary approaches to conserve threatened species are required to curb biodiversity loss. Globally, amphibians are facing the most severe declines of any vertebrate class. In response, conservation breeding programs have been established in a growing number of amphibian species as a safeguard against [...] Read more.
Multidisciplinary approaches to conserve threatened species are required to curb biodiversity loss. Globally, amphibians are facing the most severe declines of any vertebrate class. In response, conservation breeding programs have been established in a growing number of amphibian species as a safeguard against further extinction. One of the main challenges to the long-term success of conservation breeding programs is the maintenance of genetic diversity, which, if lost, poses threats to the viability and adaptive potential of at-risk populations. Integrating reproductive technologies into conservation breeding programs can greatly assist genetic management and facilitate genetic exchange between captive and wild populations, as well as reinvigorate genetic diversity from expired genotypes. The generation of offspring produced via assisted fertilisation using frozen–thawed sperm has been achieved in a small but growing number of amphibian species and is poised to be a valuable tool for the genetic management of many more threatened species globally. This review discusses the role of sperm storage in amphibian conservation, presents the state of current technologies for the short-term cold storage and cryopreservation of amphibian sperm, and discusses the generation of cryo-derived offspring. Full article
(This article belongs to the Special Issue Reproductive Biology and Assisted Reproductive Technologies in Amphibians)
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