Abstract
Embryo cryopreservation remains an important technique to enhance the reconstitution and distribution of animal populations with high genetic merit. One of the major detrimental factors to this technique is the damage caused by oxidative stress. Melatonin is widely known as an antioxidant with multi-faceted ways to counteract the oxidative stress. In this paper, we investigated the role of melatonin in protecting rabbit embryos during preimplantation development from the potential harmful effects of oxidative stress induced by in vitro culture or vitrification. Rabbit embryos at morula stages were cultured for 2 hr with 0 or 10−3 M melatonin (C or M groups). Embryos of each group were either transferred to fresh culture media (CF and MF groups) or vitrified/devitrified (CV and MV groups), then cultured in vitro for 48 hr until the blastocyst stage. The culture media were used to measure the activity of antioxidant enzymes: glutathione-s-transferase (GST) and superoxide dismutase (SOD), as well as the levels of two oxidative substrates: lipid peroxidation (LPO) and nitric oxide (NO). The blastocysts from each group were used to measure the expression of developmental-related genes (GJA1, POU5F1 and Nanog) and oxidative-stress-response-related genes (NFE2L2, SOD1 and GPX1). The data showed that melatonin promoted significantly (P<0.05) the blastocyst rate by 17% and 12% in MF and MV groups compared to their controls (CF and CV groups). The GST and SOD activity significantly increased by the treatment of melatonin in fresh or vitrified embryos, while the levels of LPO and NO decreased (P<0.05). Additionally, melatonin considerably stimulated the relative expression of GJA1, NFE2L2 and SOD1 genes in MF and MV embryos compared to CF group. Furthermore, melatonin significantly ameliorated the reduction of POU5F1 and GPX1 expression induced by vitrification. The results obtained from the current investigation provide new and clear molecular aspects regarding the mechanisms by which melatonin promotes development of both fresh and vitrified rabbit embryos.
Highlights
Livestock systems occupy about 30% of the planet's ice-free terrestrial surface area [1] and are a significant global asset with a value of at least $1.4 trillion
Previous studies mainly discussed the melatonin’s effects on the morphological variations regarding the in vitro embryo development, such as cleavage rate, blastocyst rate, hatched blastocyst rate and blastocyst cell number [26,36,52], while few studies were carried out to investigate melatonin effects at cellular and molecular levels in fresh and vitrified embryos, and rabbits were rarely used as animal models
We found that blastocyst rate significantly increased by 17% to reach 93% in the MF group compared to 76% in the compared to fresh embryos (CF) group
Summary
Livestock systems occupy about 30% of the planet's ice-free terrestrial surface area [1] and are a significant global asset with a value of at least $1.4 trillion. Overproduction of ROS induced by vitrification is detrimental for the embryos due to different types of cell injuries, including membrane lipid peroxidation, impaired intracellular milieu, disturbed metabolism, amino acid and nucleic acid oxidation, adenosine triphosphate (ATP) depletion, mitochondrial dysfunction, apoptosis and necrosis [11,12,13,14]. These negative consequences of oxidative stress suppressed gene expression involved in in vitro embryo development. Nanog gene was reported to be an important gene for pluripotency and maintaining the stem cell state in rodent embryonic stem cells [23,24]
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