Amphibian ART Over the Generations: Frozen Sperm Offspring Produce Viable F2 Generation

ORCID

CVance: https://orcid.org/0000-0001-8466-6077

MSU Affiliation

College of Agriculture and Life Sciences; Department of Biochemistry, Molecular Biology, Entomology, and Plant Pathology; College of Forest Resources; Department of Wildlife, Fisheries, and Aquaculture

Creation Date

2026-10-30

Abstract

Sperm cryopreservation is a key element in the Assisted Reproductive Technologies (ART) toolbox for stabilizing genetic diversity of captive and wild amphibian populations threatened with extinction. When expressed as spermic-urine, mature amphibian spermatozoa have characteristically long needle-shaped heads with a high proportion of surface membrane to cytosolic volume, making the cells extremely fragile and susceptible to osmotic stress. Despite these challenges to sperm cryopreservation, we have produced F1 generation offspring from cryopreserved spermic-urine in both caudates (Tiger salamander, Ambystoma tigrinum) and anurans (Mississippi gopher frog, Lithobates sevosa; Boreal toad, Anaxyrus boreas). A key question now presents itself- what is the reproductive capacity of this frozen sperm produced F1 generation? In 2015, male and female A. tigrinum were treated with human chorionic gonadotropin and gonadotropin releasing hormone for gamete development and release. Frozen sperm (5% Me2SO+0.05% BSA) thawed for IVF, yielded F1 offspring (n=21). In 2018, a mature F1 female and two males were analyzed using ultrasound for oocyte and testicular development and hormonally treated to obtain gametes. The F1 female produced 550 viable eggs; a subset(n=45) underwent IVF. Fertilization, cleavage and early embryonic development of the F2 generation to blastula stage was achieved(n=8/45;18%). The same sperm sample yielded blastula stage embryos(n=74/174; 42%) from an F0 female, control. Similarly, in 2013 spermic urine from hormonally induced L. sevosa that was frozen(10% dimethylformamide+10% trehelose) and thawed for IVF resulted in F1 offspring (26.3% frozen; 45.6% control). In 2018, spermic urine from adult F1 males and a control F0 male were used to fertilize eggs from an F0 female. The development and survivability of the F2 generation to advanced stage tadpoles/metamorphs was similar (p> 0.05) between F1 frozen males (9%, 7%) and F0 control male (8%) for the same egg mass. Thus, a frozen sperm F1 generation can produce viable gametes and offspring.

Publication Date

12-21-2018

Publication Title

Cryobiology: International Journal of Low Temperature Biology and Medicine

Publisher

Elsevier

First Page

178

Rights

© 2018 Elsevier

Link to Full Text

Share

COinS
 

Digital Object Identifier (DOI)

https://doi.org/10.1016/j.cryobiol.2018.10.220