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Theriogenology. 2016 May;85(8):1499-506. doi: 10.1016/j.theriogenology.2016.01.012. Epub 2016 Jan 19.

Viability and DNA fragmentation of rainbow trout embryos (Oncorhynchus mykiss) obtained from eggs stored at 4 °C.

Author information

1
Faculty of Medicine, Center of Biotechnology in Reproduction (CEBIOR-BIOREN), University of La Frontera, Temuco, IX Región, Chile; Faculty of Natural Resources, School of Aquaculture, Catholic University of Temuco, Temuco, IX Región, Chile. Electronic address: cubilla@proyectos.uct.cl.
2
Faculty of Natural Resources, School of Aquaculture, Catholic University of Temuco, Temuco, IX Región, Chile.
3
Faculty of Medicine, Center of Biotechnology in Reproduction (CEBIOR-BIOREN), University of La Frontera, Temuco, IX Región, Chile.
4
Faculty of Medicine, Center of Biotechnology in Reproduction (CEBIOR-BIOREN), University of La Frontera, Temuco, IX Región, Chile; Faculty of Medicine, Department of Basic Sciences, University of La Frontera, Temuco, IX Región, Chile.

Abstract

In vitro storage of salmonid eggs leads to aging of the cells causing a decline in quality and reducing their capacity to develop and produce embryos. The quality of salmonid embryos is assessed by morphologic analyses; however, data on the application of biomarkers to determine the cell viability and DNA integrity of embryos in these species are limited. The aim of this study was to evaluate the effect on embryo development, viability and DNA fragmentation in the embryonic cells of in vitro storage time at 4 °C of rainbow trout (Oncorhynchus mykiss) eggs. The embryos were obtained by IVF from eggs stored for 0 (control), 48, and 96 hours at 4 °C. At 72 hours after fertilization, dechorionated embryos were examined to determine percentages of developed embryos (embryos with normal cell division morphology), viability (LIVE/DEAD sperm viability kit), and DNA integrity (terminal deoxynucleotidyl transferase [TdT] dUTP nick-end labeling assay). The percentage of developing embryos decreased (P < 0.05) with storage time of the eggs (95.10 ± 2.55; 88.14 ± 4.50; 79.99 ± 6.60 for 0, 48, and 96 hours, respectively). Similarly, cell viability decreased (P < 0.05; 96.07 ± 7.15; 80.42 ± 8.55; 77.47 ± 7.88 for 0, 48, and 96 hours, respectively), and an increase (P < 0.05) in DNA fragmentation in the embryos was observed at 96-hour storage. A positive correlation was found between cell DNA fragmentation and storage time (r = 0.8173; P < 0.0001). The results revealed that terminal deoxynucleotidyl transferase [TdT] dUTP nick-end labeling assay technique is reliable mean to assess the state of the DNA in salmonid embryos and that in vitro eggs storage for 96h reduces embryo development and cell DNA integrity. DNA integrity evaluation constitutes a biomarker of the quality of the ova and resulting embryos so as to predict their capacity to produce good-quality embryos in salmonids, particularly under culture conditions.

KEYWORDS:

DNA; Embryo; Oocyte; Rainbow trout; Storage; Viability

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