on the experiment, COCs and spermatozoa were allowed to co-incubate for 20-24 h at 38.50C in

an atmosphere of 5% (v/v) CO2 in humidified air. After fertilization, putative zygotes were

removed from fertilization wells, denuded of cumulus cells by vortex mixing in HEPES-TALP

containing 1000 U/ml hyaluronidase, and randomly placed in groups of 25 in 50-pl drops of

either KSOM-BE2 or KSOM-BE2 containing 100 ng/mL as described previously [21]. All

drops of embryos were overlaid with mineral oil and cultured at 38.50C in an atmosphere of 5%

CO2 (experiment 1) or 5% CO2, 5% 02 and 90% N2 (experiment 2). The proportion of cleaved

oocytes was recorded on day 3 after insemination and the proportion of blastocysts and advanced

blastocysts (expanded and hatched) was recorded on day 7 (experiment 2) or day 8 (experiment

1).

Experiment 1 (Group Transfer of Embryos)

Animals

 Non-lactating Holstein cows at the University of Florida Dairy Research Unit (Hague,

FL; 29.77904 N, 82.48001 W) were used as embryo transfer recipients. Cows were kept on

pasture and supplemented with corn silage, grass hay and free-choice mineral. Animals were

synchronized for timed embryo transfer using a modified Ovsych protocol with the inclusion of a

CIDR (El-Zarhkouny et al., 2004). Cows received 100 [g of GnRH (i.m.) and a CIDR

(intravaginal deposition) on day -10. On day -3, cows received 25 mg PGF and the CIDR was

removed. A second injection of GnRH was administered on day 0 (day of anticipated ovulation).

Also on day 0, the ovaries of all cows were scanned using an Aloka 500 ultrasound (Aloka

America, Wallingford, CT) equipped with a 5 MHz linear array transducer to determine the

presence or absence of a preovulatory follicle.