Melatonin in maturation media fails to improve oocyte maturation, embryo development rates and DNA damage of bovine embryos

Abstract

Melatonin (MEL) acts as a powerful scavenger of free radicals and direct gonadal responses to melatonin have been reported in the literature. Few studies, however, have evaluated the effect of MEL during in vitro maturation (IVM) on bovine embryos. This study tested the addition of MEL to maturation medium (MM) with no gonadotropins on nuclear maturation and embryo development rates and the incidence of DNA damage in resulting embryos. Cumulus-oocyte complexes were aspirated from abattoir ovaries and cultured in MM (TCM-199 medium supplemented with 10% fetal calf serum -FCS) at 39°C and 5% CO 2 in air. After 24 hours of culture in MM with 0.5 μg mL -1 FSH and 5.0 μg mL -1 LH; 10 -9 M MEL) or 10 -9 M MEL, 0.5 μg mL -1 FSH and 5.0 μg mL -1 LH, the oocytes were stained with Hoechst 33342 to evaluate nuclear maturation rate. After in vitro fertilization and embryo culture, development rates were evaluated and the blastocysts were assessed for DNA damage by Comet assay. There was no effect of melatonin added to the MM, alone or in combination with gonadotropins, on nuclear maturation, cleavage and blastocyst rates. These rates ranged between 88% to 90%, 85% to 88% and 42% to 46%, respectively. The extent of DNA damage in embryos was also not affected by MEL supplementation during IVM. The addition of 10 -9 M MEL to the MM failed to improve nuclear maturation and embryo development rates and the incidence of DNA damage in resulting embryos, but was able to properly substitute for gonadotropins during IVM. Key words: comet assay, embryos, gonadotropins, in vitro fertilization, melatonin Melatonina no meio de maturação não melhorou as taxas de maturação dos ovócitos, de desenvolvimento embrionário e a fragmentação do DNA dos embriões bovinos RESUMO: Melatonin (MEL) atua como um potente redutor de radicais livres. Efeito direto da MEL na função gonadal também foi observado. Existem poucos estudos relacionados ao efeito da MEL durante a maturação no desenvolvimento embrionário in vitro. Avaliou-se a adição de MEL no meio de maturação (sem gonadotrofinas) nas taxas de maturação nuclear e de desenvolvimento embrionário e na incidência de fragmentação do DNA nos embriões produzidos in vitro. Complexos cumulus-ovócitos foram aspirados de ovários provenientes de matadouros e cultivados em meio de maturação (Meio TCM-199 suplementado com 10% de soro fetal bovino) a 39°C e 5% CO 2 em ar. Após 24 horas de cultivo em meio de maturação com 0,5 μg mL -1 de FSH e 5,0 μg mL -1 de LH; 10 -9 M de MEL ou 10 -9 M de MEL, 0,5 μg mL -1 de FSH e 5,0 μg mL -1 de LH, os ovócitos foram corados com Hoechst 33342 para avaliação da taxa de maturação nuclear. A fragmentação do DNA dos blastocistos foi avaliada pela técnica do Cometa. Não houve efeito da MEL adicionada ao meio de maturação, com ou sem gonadotrofinas, nas taxas de maturação nuclear, clivagem e blastocistos. Os percentuais variaram entre 88% a 90%, 85% a 88% e 42% a 46%, respectivamente. O grau de fragmentação do DNA também não foi afetado pela adição de MEL ao meio de matur...

Effects of melatonin on oocyte developmental competence and the role of melatonin receptor 1 in juvenile goats

“…Contrary to our previous results (Soto‐Heras et al, ) we did not find a higher blastocyst yield with melatonin. Other studies neither found improvement on blastocyst production (Casao et al, ; Rodrigues‐Cunha et al, ; Takada, Martins Junior, Mingoti, Balieiro, & Coelho, ) which denotes a variability in results depending on specific culture conditions. However, we observed an increase on the ICM number and ICM:TE ratio in hatched blastocysts.…”

Supplementation of IVF medium with melatonin: effect on sperm functionality andin vitroproduced bovine embryos

“…In contrast, melatonin exhibits the most desirable characteristics of a good antioxidant, scavenging a wide variety of free radicals (Galano et al, 2011). Its protective action has been described for different reproductive biotechnologies such as in vitro oocyte maturation in buffalo (Manjunatha et al, 2009) and cattle (Tsantarliotou et al, 2010;Takada et al, 2010Takada et al, , 2012Sampaio et al, 2012) improving sperm functionality in buffalo (Li et al, 2012), ram (Kaya et al, 2000;Casao et al, 2010), boar (Jang et al, 2010), mouse (Sarabia et al, 2009), hamster (Fujinoki, 2008) and human spermatozoa (Espino et al, 2010), stimulating embryo development in mice (Ishizuka et al, 2000;Gao et al, 2012), buffalo (Manjunatha et al, 2009) and cattle (Papis et al, 2007) and improving sheep embryo survival (Abecia et al, 2002). However, limited research has examined its impact on bull sperm functionality, especially during the incubation time of IVF protocol.…”

Evaluation of in vitro production rates of bovine embryos using melatonin-supplemented culture medium

“…According to Takada et al (2010) supplementation with a physiological concentration of melatonin (10 -9 M) in in vitro maturation medium with or without a combination of gonadotropins has no effect on nuclear maturation or on rates of cleavage and blastocyst formation. These rates ranged from 88% to 90%, 85% to 88%, and 42% to 46%, respectively.…”

“…The antioxidant action of melatonin is a good alternative to the in vitro production of bovine embryos. When used during the embryo maturation and cultivation processes, there are decreases in ROS and DNA fragmentation in oocytes and blastocysts (Asgari et al, 2012;Takada et al, 2010;Takada et al, 2012). However, few results have been described in the literature, especially regarding melatonin concentration to be used.…”