U/ml hyaluronidase, and randomly placed in groups of 25 in 50--l drops of either KSOM-

BE2 or KSOM-BE2 containing 100 ng/mL IGF-1 (Upstate Biotech, Lake Placid, NY) as

described previously (Block et al., 2003). All drops of embryos were overlaid with

mineral oil and cultured at 38.50C in an atmosphere of 5% CO2, 5% 02 and 90% N2. The

proportion of cleaved oocytes was recorded on d 3 after insemination and the proportion

of blastocysts and advanced blastocysts was recorded on day 7.

TUNEL Assay

 The TUNEL assay was performed as described previously (Jousan and Hansen,

2004) using an in situ cell death detection kit (Roche, Indianapolis, IN). Embryos were

removed from culture and washed two times in 50--l drops of 10 mM KPO4 pH 7.4

containing 0.9% (w/v) NaCl (PBS) and 1 mg/ml polyvinylpyrrolidone (PVP; Eastman

Kodak, Rochester, NY; PBS-PVP). Zona pellucida-intact embryos were fixed in a 50--l

drop of 4% (w/v) paraformaldehyde in PBS for 15 min at room temperature, washed

twice in PBS-PVP, and stored in 500 pl of PBS-PVP at 40C until the time of assay.

 On the day of the TUNEL assay, embryos were transferred to a 50--l drop of

PBS-PVP and then permeabilized in 0.1% (v/v) Triton X-100 containing 0.1% (w/v)

sodium citrate for 10 min at room temperature. Controls for the TUNEL assay were

incubated in 50 [l ofRQ1 RNase-free DNase (50 U/ml; Promega, Madison, WI) at 370C

in the dark for 1 h. Positive controls and treated embryos were washed in PBS-PVP and

incubated with 25 [l of TUNEL reaction mixture (containing fluorescein isothiocyanate-

conjugated dUTP and the enzyme terminal deoxynucleotidyl transferase as prepared by

and following the guidelines of the manufacturer) for 1 h at 370C in the dark. Negative

controls were incubated in the absence of terminal deoxynucleotidyl transferase. Embryos

were then washed three times in PBS-PVP and incubated in a 25-pl drop of Hoechst