Beneficial Effects of Melatonin in the Ovarian Transport Medium on In Vitro Embryo Production of Iberian Red Deer (Cervus elaphus hispanicus)

Abstract

A major limiting factor for the development of in vitro embryo production (IVP) in wild species, such as Iberian red deer, compared to livestock animals is the poor availability and limited access to biological material. Thus, the use of post-mortem ovaries from slaughtered animals represent a source of oocytes for the large scale production of embryos needed for research and to improve the efficiency of IVP. However, these oocytes are not as developmentally competent as their in vivo counterparts. Moreover, oocytes are usually obtained from ovaries that have been transported for long distances, which may also affect their quality. In order to overcome the issues associated with prolonged storage times of post-mortem material, in this study we examined the effect of melatonin supplementation to the ovary transport medium on oocyte quality, embryo yield, and blastocyst quality in Iberian red deer. When necessary, sheep was used as an experimental model due to the large number of samples required for analysis of oocyte quality parameters. Oocytes were in vitro matured and assessed for early apoptosis; DNA fragmentation; reactive oxygen species (ROS); reduced glutathione (GSH) content, mitochondrial membrane potential, and distribution; and relative abundance of mRNA transcript levels. After in vitro fertilization, embryo rates and blastocyst quality were also investigated. The results revealed that melatonin treatment significantly increased intracellular level of GSH in sheep oocytes. Moreover, the percentage of cleavage and blastocyst yield in red deer was greater compared to the Control group and there was lower abundance of oxidative stress- and apoptosis-related SHC1, TP53, and AKR1B1 mRNA transcripts in blastocysts for the Melatonin group. In conclusion, the supplementation of melatonin to the ovary storage medium had a positive effect on the developmental competence and quality of resulting blastocysts in Iberian red deer.

Cellular and Molecular Events that Occur in the Oocyte during Prolonged Ovarian Storage in Sheep

“…From those, 2327 COCs were mechanically denuded by vortex in phosphate-buffered saline (PBS) supplemented with 0.1% PVA ( w/v ; PBS-PVA) and oocytes were either directly analysed, fixed in 0.5% glutaraldehyde ( v/v ) and stored at 4 °C for terminal deoxynucleotidyl transferase mediated dUTP nick-end labelling (TUNEL) analysis or snap-frozen and stored at −80 °C for mRNA analysis. In addition, 1931 COCs, collected from the last 4 replicates, were matured, fertilized and cultured in vitro following the protocol by Sánchez-Ajofrín et al [ 10 ]. Briefly, COCs were washed in TCM199-gentamycin (4 μL/mL) and randomly placed in four-well dishes containing 500 μL of TCM199 and 4 μL/mL gentamycin, 100 μM cysteamine, 10 ng/mL follicle stimulating hormone, 10 ng/mL luteinizing hormone and 10% fetal calf serum [ 11 ] under mineral oil (Nidacon, Gothenburg, Sweden) and an atmosphere of 5% CO 2 at 38.5 °C with maximal humidity.…”