Ubilla A, Valdebenito I, Árias M E, Risopatrón J
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.
Faculty of Natural Resources, School of Aquaculture, Catholic University of Temuco, Temuco, IX Región, Chile.
Theriogenology. 2016 May;85(8):1499-506. doi: 10.1016/j.theriogenology.2016.01.012. Epub 2016 Jan 19.
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.
鲑鱼卵的体外储存会导致细胞老化，从而使卵的质量下降，并降低其发育和产生胚胎的能力。鲑鱼胚胎的质量通过形态学分析来评估；然而，关于应用生物标志物来确定这些物种胚胎的细胞活力和DNA完整性的数据有限。本研究的目的是评估虹鳟（Oncorhynchus mykiss）卵在4℃下体外储存时间对胚胎发育、活力和胚胎细胞DNA片段化的影响。通过体外受精从在4℃下储存0小时（对照）、48小时和96小时的卵中获得胚胎。在受精后72小时，对去壳胚胎进行检查，以确定发育胚胎（具有正常细胞分裂形态的胚胎）的百分比、活力（LIVE/DEAD精子活力试剂盒）和DNA完整性（末端脱氧核苷酸转移酶 [TdT] dUTP缺口末端标记试验）。随着卵的储存时间延长，发育胚胎的百分比下降（P < 0.05）（0小时、48小时和96小时分别为95.10 ± 2.55；88.14 ± 4.50；79.99 ± 6.60）。同样，细胞活力下降（P < 0.05；0小时、48小时和96小时分别为96.07 ± 7.15；80.42 ± 8.55；77.47 ± 7.88），并且在储存96小时时观察到胚胎中DNA片段化增加（P < 0.05）。发现细胞DNA片段化与储存时间之间存在正相关（r = 0.8173；P < 0.0001）。结果表明，末端脱氧核苷酸转移酶 [TdT] dUTP缺口末端标记试验技术是评估鲑鱼胚胎中DNA状态的可靠方法，并且卵在体外储存96小时会降低胚胎发育和细胞DNA完整性。DNA完整性评估构成了卵子和所得胚胎质量的生物标志物，以便预测它们在鲑鱼中产生优质胚胎的能力，特别是在养殖条件下。
