title: L-ergothioneine supplementation during culture improves quality of bovine in vitro-produced embryos.
abstract: The aim of this study was to evaluate whether supplementation of bovine culture medium with the natural antioxidant L-ergothioneine (LE), improves in vitro blastocyst development and quality, assessed as resistance to cryopreservation, total cells number, cellular differentiation, and apoptosis index. Abattoir-derived oocytes were matured and fertilized in vitro according to standard procedure. Twenty hours after IVF, presumptive zygotes were cultured in synthetic oviduct fluid with 0, 0.05 mM, 0.1 mM, 0.5 mM, and 1 mM of LE (experiment 1) at 39 °C under humidified air with 5% CO2, 7% O2, and 88% N2. On the basis of the results of this dose-response trial, the range of concentrations to test was reduced in experiment 2, in which presumptive zygotes were cultured with 0, 0.05 mM, and 0.1 mM of LE. On Day 7, embryo yields were assessed, and the blastocysts (BL) were vitrified by Cryotop method in 16.5% ethylene glycol, 16.5% DMSO and 0.5 M sucrose. Finally, BL produced on Day 8 in the absence (control) and presence of 0.1 mM LE were used for transferase-mediated dUTP nick end labeling and differential staining to evaluate, respectively the apoptotic rate and the allocation of cells into inner cell mass (ICM) and trophectoderm lineages (experiment 3). Despite similar blastocyst yields, supplementation of culture medium with 0.1 mM LE improved the cryotolerance of in vitro-produced (IVP) embryos compared to the control group, as indicated by higher (P < 0.05) hatching rates recorded after 48-hour post-warming culture (48.5%, 50.0%, and 63.8%, respectively with 0, 0.05, and 0.1 mM LE). Interestingly, when embryos were cultured in the presence of 0.1 mM LE, the percentage of BL with the most physiological ICM:total cells ratio (20%-40%) increased (85.1 vs. 66.0%, P < 0.05), confirming a beneficial effect on embryo quality. Furthermore, 0.1 mM LE decreased (P < 0.01) both the average number (4.3 ± 0.2 vs. 9.1 ± 0.3) and the proportion (3.6 ± 0.3 vs. 8.1 ± 0.5) of apoptotic cells in BL compared to the control. In conclusion, the enrichment of bovine culture medium with 0.1 mM LE improves embryo quality, as indicated by the improved cryotolerance, the lower apoptotic rate, and the higher percentage of BL with the most physiological ICM:total cells ratio.
authors.affiliation: Department of Veterinary Medicine and Animal Production, Federico II University, Naples, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Zullo
authors.affiliation: Department of Veterinary Medicine and Animal Production, Federico II University, Naples, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Albero
authors.affiliation: Department of Veterinary Medicine and Animal Production, Federico II University, Naples, Italy. Electronic address: neglia@unina.it.
authors.forename: G
authors.initials: G
authors.lastname: Neglia
authors.affiliation: Department of Veterinary Medicine and Animal Production, Federico II University, Naples, Italy.
authors.forename: C
authors.initials: C
authors.lastname: De Canditiis
authors.affiliation: Department of Veterinary Medicine and Animal Production, Federico II University, Naples, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Bifulco
authors.affiliation: Department of Veterinary Medicine and Animal Production, Federico II University, Naples, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Campanile
authors.affiliation: Department of Veterinary Medicine and Animal Production, Federico II University, Naples, Italy.
authors.forename: B
authors.initials: B
authors.lastname: Gasparrini
mesh_terms: D000818:Animals; D002417:Cattle / Q000196:embryology*; D003470:Culture Media; D046149:Embryo Culture Techniques / Q000662:veterinary*; D004624:Embryo Transfer; D004880:Ergothioneine / Q000008:administration & dosage / Q000494:pharmacology; D005307:Fertilization in Vitro / Q000662:veterinary*; D013194:Staining and Labeling
chemical_list: D003470:Culture Media; D004880:Ergothioneine
keywords: Apoptosis; Bovine; Cryotolerance; Embryo culture; L-ergothioneine