Cryoprotectants synergy improve zebrafish sperm cryopreservation and offspring skeletogenesis

Patrícia Diogo; Gil Martins; Rita Nogueira; Ana Marreiros; Paulo J Gavaia; Elsa Cabrita

doi:10.1016/j.cryobiol.2019.10.001

Cryoprotectants synergy improve zebrafish sperm cryopreservation and offspring skeletogenesis

Cryobiology. 2019 Dec:91:115-127. doi: 10.1016/j.cryobiol.2019.10.001. Epub 2019 Oct 9.

Authors

Patrícia Diogo¹, Gil Martins¹, Rita Nogueira¹, Ana Marreiros², Paulo J Gavaia³, Elsa Cabrita⁴

Affiliations

¹ Centre of Marine Sciences, University of Algarve, 8005-139, Faro, Portugal.
² Department of Biomedical Sciences and Medicine, University of Algarve, 8005-139, Faro, Portugal; Algarve Biomedical Center, Campus Gambelas, 8005-139, Faro, Portugal.
³ Centre of Marine Sciences, University of Algarve, 8005-139, Faro, Portugal; Department of Biomedical Sciences and Medicine, University of Algarve, 8005-139, Faro, Portugal.
⁴ Centre of Marine Sciences, University of Algarve, 8005-139, Faro, Portugal. Electronic address: ecabrita@ualg.pt.

PMID: 31605703
DOI: 10.1016/j.cryobiol.2019.10.001

Abstract

The synergy obtained by the combination of cryoprotectants is a successful strategy that can be beneficial on the optimization of zebrafish sperm cryopreservation. Recently, a protocol was established for this species using an electric ultrafreezer (-150 °C) performing cooling rate (-66 °C/min) and storage within one step. The ultimate objective of sperm cryopreservation is to generate healthy offspring. Therefore, the objective of this study was to select the most adequate cryoprotectant combination, for the previously established protocol, that generate high quality offspring with normal skeletogenesis. Among the permeating cryoprotectant concentrations studied 12.5% and 15% of N,N-dimethylformamide (DMF) yielded high post-thaw sperm quality and hatching rates. For these two concentrations, the presence of bovine serum albumin (10 mg/mL), egg yolk (10%), glycine (30 mM) and bicine (50 mM) was evaluated for post-thaw sperm motility, viability, in vitro fertilization success and offspring skeletal development (30 days post fertilization). Higher concentration of permeating cryoprotectant (15%) decreased the incidence of deformed arches and severe skeletal malformations, which suggests higher capacity to protect the cell against cold stress and DNA damage. Extender containing 15% DMF with Ctrl, Bicine and egg yolk were the non-permeating cryoprotectants with higher post-thaw quality. The use of these compounds results in a reduction in vertebral fusions, compressions and severity of skeletal malformations in the offspring. Therefore, these extender compositions are beneficial for the quality of zebrafish offspring sired by cryopreserved sperm with -66 °C/min freezing rate. To the best of our knowledge, this is the first report on skeletal development of the offspring sired by cryopreserved sperm performed with different freezing media compositions in zebrafish.

Keywords: Cryopreservation; Cryoprotectants; Decision trees; Offspring; Skeletal malformations; Zebrafish sperm.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Albumins / pharmacology
Animals
Cryopreservation / methods*
Cryoprotective Agents / pharmacology*
Dimethylformamide / pharmacology*
Egg Yolk
Freezing
Glycine / analogs & derivatives
Glycine / pharmacology
Male
Semen Preservation / methods*
Sperm Motility / drug effects
Spermatozoa / drug effects
Zebrafish / embryology*

Substances

Albumins
Cryoprotective Agents
N,N-bis(2-hydroxyethyl)glycine
Dimethylformamide
Glycine