3 g mL of luteinizing hormone that was generously provided by the USDA, Beltsville, MD, 10% FBS, 0. 2 M sodium pyruvate and 2 mM glutamine. Mod ified Tyrodes Albumin Lactate Pyruvate media used in sperm preparation, removal of cumu lus cells from oocytes and in vitro fertiliza tion were prepared as described by Parrish selleckchem Tofacitinib et al. In vitro culture medium was a modified synthetic oviductal fluid supplemented with 3 mg mL of BSA, 0. 6 mM sodium pyruvate, 2% BME essential amino acids, 1% MEM non essential amino acids, and 100 g mL of penicillin and streptomycin. All trans retinol was dissolved in 100% ethanol, appropri ate dilutions made, and aliquots were stored at 80 C until use. Retinol was prepared fresh each month and checked on a spectrophotometer for accuracy.

The con centration of ethanol during maturation or culture was less than 0. 1%. Collection and in vitro maturation of oocytes Ovaries from mature, cycling cattle were obtained from an abbatoir and pooled. Cumulus oocyte comple es were quickly harvested by slicing follicles with a sterile surgical blade, and collecting them in OCM. Intact COCs with homogeneous ooplasm and two or more layers of cumulus cells were selected, washed, and appro imately 50 were transferred to 500 l of pre equil ibrated OMM, and matured for 22 23 hours in a 38. 5 C incubator with an atmosphere of 5. 0% CO2, ambient air, and saturated humidity. In vitro fertilization Fertilization was performed with combined semen from two bulls of proven fertility prepared accord ing to the method by Parrish and coworkers.

Briefly, spermatozoa were washed in a discontinuous Percoll gra dient by depositing semen on top of the Per coll layers and centrifuged for 15 minutes at 960 g. The pellet was removed and resuspended in SP TALP and cen trifuged for 8 minutes at 460 g. After removal of the super natant, the sperm sample was reconstituted in 500 L of IVF TALP for a final concentration of 1 106 spermato zoa mL. The plate was incubated for 22 hours at 38. 5 C in an atmosphere of 5. 0% CO2 and ambient air with satu rated humidity. In vitro culture Appro imately 18 hours after fertilization putative zygotes were denuded of cumulus cells by vorte ing in 500 l of HEPES TALP for four minutes. Putative zygotes were cultured in 500 L of mSOF for eight days in a 38. 5 C incubator in an atmos phere of 5% CO2, 7% O2 and 88% N2 with saturated humidity.

The mSOF medium was changed every 48 hours. Cleavage was assessed on Day 3 and blastocyst rate was calculated on Day 8. E perimental Design In the first e periment maturation medium Drug_discovery alone was supplemented with all trans retinol and embryos were allowed to develop under low o y gen conditions. In the second e periment all trans retinol was added only to embryo culture medium on days 1, 3, 5, and 7, and the embryos developed in a low o ygen atmosphere.