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88 enrichment of culture medium with crocetin improves in vitro embryo development in cattle

Zullo, G., Tamayo Palacio, J. E., De Canditiis, C., Longobardi, V., Salzano, A., Albero, G., Gasparrini, B.
Reproduction, fertility, and development 2016 v.28 no.2 pp. 173
air, analysis of variance, animal reproduction, antioxidant activity, blastocyst, carbon dioxide, cattle, culture media, developmental stages, dose response, embryogenesis, enrichment culture, in vitro fertilization, oocytes, oxidative stress, oxygen, saffron, viability, zygote
The high incidence of developmental failure of bovine in vitro-produced embryos is due to suboptimal culture conditions that induce oxidative stress. Indeed, increased oxidative stress is one of the main factors affecting in vitro mammalian embryo development, decreasing the viability of IVP embryos. It is known that saffron has a powerful antioxidant capacity, mainly due to its active components crocin and crocetin. The aim of this study was to evaluate whether enriching the in vitro culture medium with crocetin improves in vitro embryo production efficiency in cattle. The range of concentrations of crocetin was chosen after a preliminary dose response trial (322 total presumptive zygotes were cultured with 0, 1, 10, and 50μM, over 2 replicates) that showed beneficial and deleterious effects, respectively, with the lowest and highest concentration compared with the control (36.6±5.6, 57.4±4.5, 46.4±4.4, and 6.8±3.7% blastocyst rates, respectively, with 0, 1, 10, and 50μM; P<0.01). Therefore, the range of concentrations to test was reduced. Abattoir-derived bovine oocytes (n=832, over 4 replicates) were in vitro matured and fertilized according to standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347–1355). Twenty hours after IVF, presumptive zygotes were cultured in SOF medium with 0 (control; n=208), 1μM (n=208), 2.5μM (n=208), and 5μM (n=208), at 39°C under humidified air with 5% CO2, 7% O2, and 88% N2. The embryos obtained by the end of culture (i.e. on Day 7 post-IVF) were scored for quality, based on morphological criteria, and for developmental stage, as previously described (Robertson and Nelson 2010, Manual of the IETS, 86–105). The percentages of total transferable embryos and grade 1 and 2 blastocysts were recorded. As the chronology of development is a reliable parameter to assess quality, the percentage of fast-developing embryos (i.e. hatched and expanded blastocysts) was also compared among groups. Differences among groups were analysed by ANOVA, and Tukey method was used as a post-hoc test. Data are presented as means±s.d. The supplementation of crocetin during culture did not affect cleavage rate (74.9±6.3, 76.4±8.4, 81.4±4.3, and 76.4±8.4%, respectively, with 0, 1, 2.5, and 5μM). However, post-fertilization embryo development improved with 1µM crocetin compared with the control, both in terms of total embryo output (43.8±4.4, 61.1±5.2, 50.4±6.7, and 53.3±7.3%, respectively, with 0, 1, 2.5, and 5μM; P<0.01) and grade 1 and 2 blastocysts (41.0±3.6, 54.3±5.4, 46.2±6.7, and 49.4±6.5%, respectively, with 0, 1, 2.5, and 5μM; P<0.05), whereas no differences were observed among the other groups. Moreover, the percentage of fast developing embryos increased with 1µM (P<0.05) crocetin compared with the control, with no other differences recorded among groups (17.7±5.8, 34.7±5.7, 24.9±5.1, and 28.7±7.8%, respectively, with 0, 1, 2.5, and 5μM). In conclusion, these results demonstrated a beneficial effect of low concentrations of crocetin (1μM) during culture both on blastocyst yield and quality, as indicated by the improved chronology of embryo development.