title: Crocetin improves the quality of in vitro-produced bovine embryos: Implications for blastocyst development, cryotolerance, and apoptosis.
abstract: The aim of this work was to assess the effect of supplementation of bovine culture medium with the natural antioxidant crocetin on in vitro blastocyst development and quality. This was evaluated as cryotolerance, apoptosis index, and total cells number and allocation. 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 medium, supplemented with 0, 1, 2.5, and 5 μM crocetin (experiment 1) at 39 °C under humidified air with 5% CO2, 7% O2, and 88% N2. On Day 7, embryo yields were assessed and the blastocysts were vitrified by Cryotop method in 16.5% ethylene glycol, 16.5% DMSO, and 0.5 M sucrose. Finally, blastocysts produced on Day 8 in the absence (control) and presence of 1 μM crocetin were used for terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling and differential staining to evaluate, respectively, the apoptotic rate and the allocation of cells into inner cell mass (ICM) and trophectoderm (TE) lineages (experiment 2). Embryo development was higher in the 1 μM crocetin group compared to the control, both in terms of total embryo output (37.7 ± 4.2%, 52.9 ± 6.3%, 40.9 ± 7.6%, and 42.4 ± 8.7%, respectively, with 0, 1, 2.5, and 5 μM; P < 0.01) and grade 1 and 2 blastocysts (33.6 ± 4.9%, 46.1 ± 7.3%, 37.8 ± 7.9%, and 39.4 ± 7.9%, respectively, with 0, 1, 2.5, and 5 μM; P < 0.05). Moreover, the percentage of fast-developing embryos increased in 1 μM crocetin group compared to the control (23.4 ± 4.7%, 32.7 ± 6.6%, 27.2 ± 6.6%, and 30.1 ± 7.2%, respectively, with 0, 1, 2.5, and 5 μM; P < 0.05). In addition, the enrichment of culture medium with 1 μM crocetin improved embryo cryotolerance compared to the control, as indicated by higher hatching rates recorded after 48 hours postwarming culture (46.5% vs. 60.4%; P < 0.05). Furthermore, 1 μM crocetin decreased both the average number (9.9 ± 0.4 vs. 7.1 ± 0.3) and the percentage of apoptotic cells (7.1 ± 0.4 vs. 4.2 ± 0.2) in blastocysts compared to the control (P < 0.01). However, no differences were recorded in the average number of ICM, TE, and total cells between 1 μM crocetin and control groups. In conclusion, the enrichment of bovine culture medium with 1 μM crocetin increased both blastocyst yield and quality, as indicated by the improved chronology of embryo development, increased resistance to cryopreservation, and reduced incidence of apoptosis.
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: C
authors.initials: C
authors.lastname: De Canditiis
authors.affiliation: Department of Veterinary Medicine and Animal Production, Federico II University, Naples, Italy.
authors.forename: M E
authors.initials: ME
authors.lastname: Pero
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.
authors.forename: A
authors.initials: A
authors.lastname: Salzano
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: 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; D017209:Apoptosis; D001755:Blastocyst / Q000187:drug effects* / Q000502:physiology; D002338:Carotenoids / Q000494:pharmacology*; D002417:Cattle / Q000196:embryology*; D015925:Cryopreservation / Q000662:veterinary*; D046149:Embryo Culture Techniques / Q000662:veterinary*; D047108:Embryonic Development / Q000187:drug effects; D014801:Vitamin A / Q000031:analogs & derivatives
chemical_list: C487773:trans-sodium crocetinate; D014801:Vitamin A; D002338:Carotenoids
keywords: Apoptosis; Bovine; Crocetin; Cryotolerance; Embryo culture