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.

Keywords: deer; embryo; melatonin; oocyte; ovary storage; sheep; transport.

Figure 1

Representative images of early apoptosis detection in sheep oocytes. Oocytes were stained by Annexin V and PI. (A) Viable oocytes; (B) early apoptotic oocytes; (C,D) non-viable oocytes. Scale bar = 52 µM.

Figure 2

Representative images of DNA fragmentation by TUNEL assay in sheep oocytes at the germinal vesicle (GV) stage. (A) TUNEL-positive oocyte; (B) TUNEL-negative oocyte. Scale bar = 38 µM.

Figure 3

Representative images of reactive oxygen species (ROS) and glutathione (GSH) levels in sheep oocytes. (A) High ROS intensity; (B) low ROS intensity; (C) high GSH intensity; (D) low GSH intensity. Scale bar = 51 µM.

Figure 4

Representative images of JC-1 stained mitochondria in sheep oocytes. (A) Mitochondria with high membrane potential (red fluorescence); (B) mitochondria with low membrane potential (green fluorescence). Scale bar = 21 µM.

Figure 5

Representative images of mitochondrial distribution of sheep oocytes. (A) Red mitochondria normally distributed (homogeneous distribution; Figure 5A); (B) red mitochondria abnormally distributed (Figure 5B). Scale bar = 24 µM.

Figure 6

Melatonin concentrations (pg/mL) in samples of follicular fluid collected from sheep ovaries transported in saline solution (Control) and saline solution with 10⁻³ M melatonin (Melatonin); Results are expressed as mean ± SEM; ^a,b Different letters indicate differences (p ≤ 0.05) among treatments.

Figure 7

Effect of melatonin supplementation on sheep oocytes’ (A) early apoptosis (positive Annexin V staining) and DNA fragmentation (positive TUNEL staining); (B) ROS and GSH levels; (C) homogeneous mitochondrial distribution; (D) mitochondrial membrane potential: red (high membrane potential)/green (low membrane potential) fluorescence ratio (JC-1 staining); Results are expressed as mean ± SEM; ^a,b Different letters indicate differences (p ≤ 0.05) among treatments.

Figure 8

Relative mRNA transcript abundance in Iberian red deer oocytes collected from ovary stored with (Melatonin) and without melatonin (Control); Results are expressed as mean ± SEM.

Figure 9

Relative mRNA transcript abundance in Iberia red deer blastocysts produced in vitro from ovary stored with (Melatonin) and without melatonin (Control); Results are expressed as mean ± SEM; ^a,b Different letters indicate differences (p ≤ 0.05) among treatments.