288 PRELIMINARY RESULTS OF THE USE OF TUMOR CELLS OF A DOG TO CONDITION BOVINE EMBRYO IN VITRO CULTURE MEDIUM

Abstract

The aim of this study was to analyze the response of bovine embryos fertilized in vitro to a tumor dog cell- conditioned TCM-199 culture medium used in the development stage. Two hundred ninety-five oocytes obtained from slaughterhouse ovaries were cultured in groups of 20 cells at 39°C in TCM-199 medium droplets with 20% estrous cow serum (ECS) for 24h in a humid atmosphere containing 5% CO2. After incubation, these oocytes were fertilized with a 1 × 106 sperm cell dose, previously capacitated by a 1-h incubation in SP-TALP medium under usual conditions, by incubation in small petri dishes with 1 mL of Fert-TALP medium for 24 h under the conditions described previously. Alleged fertilized oocytes were randomly separated into 2 groups. A control group (n = 144) was cultured in TCM-199 medium with 10% fetal calf serum (FCS) and the other (n = 151) was cultured in TCM-199 medium previously conditioned by incubation for 72 h with 1 × 106 cells of a complex adenocarcinoma grade 1 obtained from a 6-year-old boxer dog and sterilized by filtration with 0.2-(im membrane. After 72 h of culture, the zygotes were harvested and prepared for a quick microscope observation. Alleged zygotes were classified as “not fertilized,” “divided,” or “degenerated.” Statistical differences (P < 0.01) were found among both groups with a greater number of normal divided embryos (65.2%) in the conditioned medium group and a lower number (30.4%) in the control group. The number of normal divided embryos in the conditioned medium after 72 h of incubation was similar to that achieved by current methods. These preliminary results are consistent with results obtained by other researchers in which growth factors derived from established cell lines can be used to condition culture medium for in vitro bovine embryo development. Further studies will be necessary to clarify how these factors contribute to in vitro embryo development. Spanish Retinta Breed Asociation, Spain.