148 AN EFFECT OF MELATONIN ON DEVELOPMENT OF BOVINE EMBRYOS CULTURED IN VITRO UNDER OPTIMAL OR ENHANCED OXYGEN TENSIONS

Abstract

Many different systems of free radical scavengers have been investigated during the last few years for in vitro culture of mammalian embryos. Melatonin is a potent reactive oxygen species scavenger and has been tested in the promotion of mouse embryo development in vitro (Ishizuka et al. 2000 J. Pin. Res. 28, 48–51). An effect of melatonin on bovine embryo development in vitro is described here. Slaughterhouse-derived oocytes were subjected to standard in vitro maturation and fertilization procedures. Presumptive zygotes randomly allocated to experimental groups were cultured for 3 days (Day 1–Day 3) in CR1aaLA medium (Papis et al. 2000 Theriogenology 54, 651–658) supplemented with two different concentrations of melatonin (10⁻⁶ M or 10⁻⁴ M; Sigma, St. Louis, MO, USA) or without melatonin (control). Culture was performed under two different gas atmospheres containing 4% CO₂ and either normal (7%) or enhanced (20%) oxygen concentration (2 × 3 factorial analysis). At the end of Day 3, embryos from each treatment group, developed to at least the 4-cell stage, were collected and cultured without melatonin until Day 10 at optimum 4% CO₂ and 7% O₂ atmosphere. The numbers of blastocysts at Day 8 and hatching/hatched blastocysts at Day 10 were recorded. Five replicates of each treatment were performed. Blastocyst formation rates of presumptive zygotes and of Day 3, 4-cell embryos were calculated for each group. Differences between groups were analyzed using chi-square and/or Fisher's exact tests where appropriate. P < 0.05 was considered statistically significant. Out of 100, 100, and 101 presumptive zygotes cultured for the first 3 days in 7% oxygen with 10⁻⁴ M, 10⁻⁶ M, or no melatonin, 31 (31%), 40 (40%), and 44 (43.5%) developed to blastocyst stage and 25 (25%), 33 (33%), and 36 (36%) to hatching/hatched blastocyst stage, respectively. On the other hand, out of 102, 102, and 100 zygotes cultured in the same concentrations of melatonin, but under 20% of oxygen, an opposite tendency was observed, as 42 (41%), 25 (24.5%), and 32 (32%) blastocysts and 26 (25.5%), 21 (20.6%), and 25 (25%) hatching/hatched blastocysts developed, respectively. No statistical significance was reached here. However, out of 4-cell embryos put into in vitro culture after initial treatments in different melatonin concentrations, a decreased ratio of blastocyst formation was observed in the 10⁻⁴ M melatonin group (31/65, 47.7%) compared to that of the control (44/65, 67.7%; P = 0.0327) when the lower oxygen concentration was applied. However, a beneficial effect of melatonin was observed in the presence of 20% oxygen. Out of 61 embryos, 42 (68.9%) developed to the blastocyst stage after treatment in 10⁻⁴ M melatonin concentrations, vs. 32/63 (50.8%; P = 0.0458) blastocysts developed in control group. In conclusion, a beneficial or a harmful effect of melatonin on bovine embryo in vitro development was observed depending on the oxygen concentration during the treatment. Results presented seem to confirm a potent free radicals scavenging activity of melatonin in a bovine embryo culture system.