Natural antioxidants during in vitro culture improve embryo quality in cattle

In cattle, in vitro-produced (IVP) bovine embryos have been around in large numbers for over two decades. It is probably fair to say that their main use has been in research, as a means of understanding what happens during normal in vivo embryo development in cattle, as stepping stones to other technologies, such as nuclear transfer, and as a tool for examining what goes wrong in development following such procedures, and as models from which to extrapolate findings to other species. The high incidence of developmental failure in the in vitro embryo production system has been also attributed to suboptimal culture conditions that induce oxidative stress. Oxidative stress has been implicated in many different types of cell injuries, including membrane lipids peroxidation, oxidation of amino acids and nucleic acids, apoptosis and necrosis which may subsequently decrease the viability of IVP embryos. To overcome this, exogenous antioxidant compounds have been frequently used to increase antioxidant capacity of embryos via increasing intracellular levels of reactive oxygen species (ROS) scavengers. Development of accurate laboratory methods to assess embryo quality will improve the efficiency of embryo production from in-vitro culture systems. Currently, the techniques of TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL) for the detection of apoptosis, and differential staining for determining the ratio of inner cell mass (ICM) to trophectoderm (TE) cells, are used separately to assess embryo quality in a range of different species. Furthermore, the ability to cryopreserve embryos, without critical loss of viability, has a profound effect on the success of assisted reproductive techniques. The survival of cryopreserved in vitro produced (IVP) embryos, as measured either by post-warming survival in culture or by established pregnancies after embryo transfer, has lagged behind that of in vivo-derived embryos. Even if the background of this difference is not completely understood, it is known that the culture medium has dramatic effect on developmental competence and cryo-withstand of the resulting embryos. The aim of this research was to evaluate whether supplementation of bovine culture medium with different natural antioxidant compounds such as Resveratrol, L-Ergothioneine and Crocetin, found in several vegetal sources, improves in vitro blastocyst development and quality, assessed as resistance to cryopreservation, total cells number, ratio of ICM:TE cells and apoptosis index. Abattoir-derived oocytes were matured and fertilized in vitro according to our standard procedure. Twenty h after IVF, presumptive zygotes were cultured in SOF medium, supplemented with different concentrations of each antioxidant compound such as resveratrol (0, 0.25, 0.5 and 1 µM), L-ergothioneine (0, 0.05, 0.1 and 0.5 mM) and Crocetin (0, 1, 2.5 and 5 µM) at 39°C under humidified air with 5% CO2, 7% O2 and 88% N2. On Day 7 (IVF = Day 0) embryo yields were assessed and the blastocysts were vitrified by cryotop method in 16.5% ethylene glycol, 16.5% dimethyl sulfoxide (DMSO) and 0.5 M sucrose. Finally, blastocysts produced on day 8 in the absence (control) and presence of 0.5 µM resveratrol, 0.1mM L-ergothioneine and 1 µM Crocetin were used for TUNEL and differential staining to evaluate respectively the apoptotic rate and the allocation of cells into ICM and TE lineages. The results showed that: Resveratrol supplementation did not increase blastocyst yields, but 0.5 µM resveratrol improved the cryotolerance of IVP embryos compared to the control, as indicated by higher development rates and hatching rates recorded after 48 h post-warming culture. Blastocysts produced in the absence (control) and presence of 0.5 µM resveratrol had a similar number of ICM, TE and total cells, as well a similar ratio of ICM:total cells. L-ergothioneine supplementation did not increase blastocyst yields, but 0.1 mM L-ergothioneine group showed a better cryotolerance of IVP embryos compared to the control group, as indicated by higher hatching rates recorded after 48 h post-warming culture. In addition, blastocysts produced in the presence of 0.1 mM L-ergothioneine have proved a higher embryo quality compared to the control, as indicated by lower percentage of apoptotic rate and higher percentage of blastocysts with the most physiological ICM:total cells ratio. In contrast to previous antioxidant compounds, Crocetin supplementation of 1 µM increase blastocyst yields in term of higher total embryo output, superior quality blastocysts and fast developing embryos percentages. In addition, this crocetin concentration improved the cryotolerance of IVP embryos compared to the control, as indicated by higher hatching rates recorded after 48 h post-warming culture. Blastocysts produced in the absence (control) and presence of 1 µM crocetin had a similar number of ICM, TE and total cells, as well a similar ratio of ICM:total cells. Furthermore, blastocysts produced in IVC medium enriched with 1 µM crocetin have proved a higher embryo quality compared to the control, showing a lower percentage of apoptotic rate.