Morphological evaluation of Prochilodus lineatus embryos after vitrification–thawing in high‐osmolarity cryoprotectant solution. (16th July 2018)

• Record Type: Journal Article
• Authors: Costa, Raphael da Silva; Capuzzo, Caio de Souza; Ribeiro, Cristiele da Silva; Verissimo‐Silveira, Rosicleire; Siqueira‐Silva, Diógenes Henrique de; Senhorini, José Augusto; Ninhaus‐Silveira, Alexandre
• Is Part Of: Reproduction in domestic animals. Volume 53:Number 6(2018)
• Page Start: 1353
• Page End: 1358
• Publication Date: 2018-07-16
• Subjects: cryopreservation -- cryoprotectant -- ice -- injuries -- recrystallization -- vitrification; Animal breeding -- Periodicals; Veterinary pathology -- Periodicals; Livestock -- Breeding -- Periodicals; 636.082
• DOI: 10.1111/rda.13249
• Languages: English
• ISSNs: 0936-6768

Abstract

Contents: We aimed to vitrify embryos of Prochilodus lineatus in a high‐osmolarity cryoprotectant solution, evaluating, after the vitrification–thawing process, their morphological changes. Thus, 240 embryos in the 20‐somite phase (20S) were exposed for 20 min to one main internal cryoprotectant solution (1, 2‐propanediol—PROP), divided into four immersion sequence steps of five minutes each. The first three steps were performed in solutions containing only a main internal cryoprotectant (PROP—2, 3 and 4 M), and the fourth step in a high‐osmolarity solution combining internal (PROP + dimethyl sulphoxide—Me2 SO) and external cryoprotectants (sucrose—SUC). The final concentration of vitrification was PROP 5 M + Me2 SO 5 M + SUC 0.2 M. During vitrification, the straws exhibited a translucent solid appearance; however, during thawing, their structure became totally opaque and white. After thawing, the embryos suffered an increase in volume and presented morphological changes including protrusions on the surface of the yolk sac, yolk sac rupture, and optical vesicle degradation. On the inside, we observed intercellular spaces and a yolk syncytial layer (YSL) with altered chromatin. Yet, structures such as somites, neural tube, endoderm and epidermis presented cells with a nucleus and integral mitochondria. We conclude that the use of the tested cryoprotectant solution permits the formation of a vitreous solid and preserves part of the cells of the blastoderm. Yet, the heating protocol does not control recrystallization, resulting in the formation of serious morphological anomalies that prevent the preservation of the embryonic unit.