title: Cell apoptosis and lipid content of in vitro-produced, vitrified bovine embryos treated with forskolin.
abstract: The presence of fetal calf serum in culture medium influences embryo quality, causing a reduction in postcryopreservation survival. Forskolin has been used to induce lipolysis and increase cryotolerance, functioning as an activator of adenylate cyclase and elevating cAMP levels. In the present experiment, bovine zygotes were cultured in synthetic oviduct fluid with amino acid plus 2.5% fetal calf serum for 6 days, when forskolin was added in three concentrations: 2.5, 5, and 10 μM. Treatment with forskolin lasted for 24 hours. Blastocyst formation rate, quantification of lipid granules, total cell numbers, and apoptosis rate were evaluated. In a second assessment, embryos were vitrified, and warming, re-expansion rate, total cell numbers, and apoptosis rate were also evaluated. There was no difference due to forskolin in blastocyst formation or re-expansion rates after vitrification. However, lipid measurements were lower (control: 136.8 and F 2.5 μM: 128.5; P < 0.05), and number of cells per embryo higher (control: 140.1 and F 2.5 μM: 173.5; P < 0.05) than controls for 2.5 μM forskolin but not for higher forskolin concentrations. The number of intact cells per embryo was higher, and the rate of apoptosis was lower in fresh than in vitrified embryos (number of cells of warmed embryos, control: 104.1, F 2.5 μM: 101.3, F 5 μM: 115.4, F 10 μM: 95.1; apoptotic of fresh cells, control: 12.1%, F 2.5 μM: 16.7%, F 5 μM: 11.1%, F 10 μM: 14.2%; and apoptotic warmed embryos, control: 22.3%, F 2.5 μM: 37.3%, F 5 μM: 33.2%, F 10 μM: 30.3%; P < 0.05). It was concluded that forskolin is an effective lipolytic agent even at low concentrations, leading to formation of blastocysts with a comparatively larger number of cells.
authors.affiliation: Departmento de Medicina Veterinária, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, Pirassununga, SP, Brazil. Electronic address: dmpaschoal@gmail.com.
authors.forename: Daniela Martins
authors.initials: DM
authors.lastname: Paschoal
authors.affiliation: Universidade Federal do Pampa, Faculdade de Medicina Veterinária, Uruguaiana, RS, Brazil.
authors.forename: Mateus José
authors.initials: MJ
authors.lastname: Sudano
authors.affiliation: Departmento de Medicina Veterinária, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, Pirassununga, SP, Brazil.
authors.forename: Kátia Regina Lancellotti
authors.initials: KRL
authors.lastname: Schwarz
authors.affiliation: Departmento de Reprodução Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade do Estado de São Paulo, Botucatu, SP, Brazil.
authors.forename: Rosiára Rosário Dias
authors.initials: RRD
authors.lastname: Maziero
authors.affiliation: Departmento de Reprodução Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade do Estado de São Paulo, Botucatu, SP, Brazil.
authors.forename: Midyan Daroz
authors.initials: MD
authors.lastname: Guastali
authors.affiliation: Departmento de Reprodução Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade do Estado de São Paulo, Botucatu, SP, Brazil.
authors.forename: Letícia Ferrari
authors.initials: LF
authors.lastname: Crocomo
authors.affiliation: Departmento de Reprodução Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade do Estado de São Paulo, Botucatu, SP, Brazil.
authors.forename: Luis Carlos Oña
authors.initials: LCO
authors.lastname: Magalhães
authors.affiliation: Departmento de Clínica, Cirurgia e Reprodução Animal, Faculdade de Medicina Veterinária, Universidade do Estado de São Paulo, Araçatuba, SP, Brazil.
authors.forename: Alício
authors.initials: A
authors.lastname: Martins
authors.affiliation: Departmento de Medicina Veterinária, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, Pirassununga, SP, Brazil.
authors.forename: Claudia Lima Verde
authors.initials: CLV
authors.lastname: Leal
authors.affiliation: Departmento de Reprodução Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade do Estado de São Paulo, Botucatu, SP, Brazil.
authors.forename: Fernanda da Cruz
authors.initials: FDC
authors.lastname: Landim-Alvarenga
mesh_terms: D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002417:Cattle / Q000196:embryology*; D005576:Colforsin / Q000494:pharmacology*; D015925:Cryopreservation / Q000662:veterinary*; D005307:Fertilization in Vitro / Q000662:veterinary*; D059471:In Vitro Oocyte Maturation Techniques / Q000662:veterinary; D008055:Lipids / Q000737:chemistry*; D058989:Vitrification
chemical_list: D000276:Adjuvants, Immunologic; D008055:Lipids; D005576:Colforsin
keywords: Apoptosis; Blastocyst; Cryopreservation; Forskolin; Lipid; Vitrification