Skim milk powder used as a non-permeable cryoprotectant reduces oxidative and DNA damage in cryopreserved zebrafish sperm

Rômulo Batista Rodrigues; Mariana Uczay; Verônica Bidinotto Brito; Ana Amélia Nunes Fossati; Antonio Cesar Godoy; Dinara Jaqueline Moura; Carla Ivane Ganz Vogel; Ana Carina Nogueira Vasconcelos; Danilo Pedro Streit Jr

doi:10.1016/j.cryobiol.2020.10.005

Skim milk powder used as a non-permeable cryoprotectant reduces oxidative and DNA damage in cryopreserved zebrafish sperm

Cryobiology. 2020 Dec:97:76-84. doi: 10.1016/j.cryobiol.2020.10.005. Epub 2020 Oct 7.

Authors

Rômulo Batista Rodrigues¹, Mariana Uczay², Verônica Bidinotto Brito³, Ana Amélia Nunes Fossati⁴, Antonio Cesar Godoy⁵, Dinara Jaqueline Moura⁶, Carla Ivane Ganz Vogel⁷, Ana Carina Nogueira Vasconcelos⁸, Danilo Pedro Streit Jr⁹

Affiliations

¹ Graduate Degree Program in Animal Science, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil. Electronic address: [email protected].
² Graduate Degree Program in Pharmacology and Therapeutics, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil. Electronic address: [email protected].
³ Department of Physiotherapy, Integrated Faculties of Taquara, Taquara, RS, Brazil; Laboratory of Toxicological Genetics, Postgraduate Program in Health Sciences, Federal University of Health Sciences of Porto Alegre, Porto Alegre, RS, Brazil. Electronic address: [email protected].
⁴ Graduate Degree Program in Animal Science, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil. Electronic address: [email protected].
⁵ Department of Chemistry, State University of Maringá, Maringá, PR, Brazil. Electronic address: [email protected].
⁶ Laboratory of Toxicological Genetics, Postgraduate Program in Health Sciences, Federal University of Health Sciences of Porto Alegre, Porto Alegre, RS, Brazil. Electronic address: [email protected].
⁷ Biochemistry Laboratory, Multicenter Graduate Degree Program in Biochemistry and Molecular Biology, University of Santa Catarina State, Lages, SC, Brazil. Electronic address: [email protected].
⁸ Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD, USA. Electronic address: [email protected].
⁹ Graduate Degree Program in Animal Science, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil. Electronic address: [email protected].

PMID: 33038414
DOI: 10.1016/j.cryobiol.2020.10.005

Abstract

Cryoprotectants play a vital role in the cryopreservation process, protecting biological samples from freezing damage. Here, we evaluate the effects of the combination and interaction of different extenders with permeable and non-permeable cryoprotectants, on the cryopreservation of Danio rerio sperm, analyzing the effects of cryopreservation through a broad approach to variables. Two extenders were used, Hank's balanced salt solution (HBSS) and Ginsburg's solution. Eight cryoprotective solutions (CS) were used: CS1 (HBSS + Me₂SO 8%), CS2 (HBSS + Methanol 8%), CS3 (HBSS + Me₂SO 8% + Skim milk powder 15%), CS4 (HBSS + Methanol 8% + Skim milk powder 15%), CS5 (Ginsburg + Me₂SO 8%), CS6 (Ginsburg + Methanol 8%), CS7 (Ginsburg + Me₂SO 8% + Skim milk powder 15%) and CS8 (Ginsburg + Methanol 8% + Skim milk powder 15%). The samples were cryopreserved in cryovials for 20 min on dry ice, stored in liquid nitrogen, thawed at 38 °C for 10 s, and analyzed. In addition to increasing viability, we show that powdered milk also allows for better preservation of the membrane and normal cell morphology, and protects the sperm cells from DNA damage and oxidative stress caused by cryopreservation.

Keywords: Cell oxidation; Comet assay; Cryoinjury; DNA fragmentation; Danio rerio.

Publication types

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

MeSH terms

Animals
Cryopreservation* / methods
Cryoprotective Agents / pharmacology
DNA Damage
Dimethyl Sulfoxide
Male
Milk
Oxidative Stress
Powders
Semen Preservation*
Sperm Motility
Spermatozoa
Zebrafish

Substances

Cryoprotective Agents
Powders
Dimethyl Sulfoxide