| Summary: | <i>In ovo</i> injection of the Marek’s disease (MD) vaccine (MDV) has been widely practiced in commercial US hatcheries. However, the MDV is very sensitive and may not be compatible with some nutrients when administered together by <i>in ovo</i> injection. When individually administered by <i>in ovo</i> injection, L-Ascorbic acid (L-AA) and 25-hydroxyvitamin D<sub>3</sub> (25OHD<sub>3</sub>) have previously exhibited very promising results on the post-hatch physiological and immunological characteristics of broilers subjected to stressful commercial conditions. However, the compatibility of the MDV with these vitamins has not been previously explored. Their compatibility must first be established before their combined administration by <i>in ovo</i> injection can be considered. Therefore, the objective in this study was to determine the compatibility of the MDV with various levels of 25OHD<sub>3</sub> or L-AA. The treatments employed were MDV-alone, MDV in combination with 0.6 (low) or 2.4 (high) μg doses of 25OHD<sub>3</sub>, or MDV in combination with 1.2 (low) or 12 (high) mg doses of L-AA. The live and dead ratio of primary chick embryo fibroblast cells infected by the MD virus (CEF-MDV) in each treatment was determined every 30 min for 2 h. The L-AA at both the low and high doses resulted in a 70% death of CEF-MDV within 1 h, but either dose of the 25OHD<sub>3</sub> exhibited only an approximate 5% lower CEF-MDV survival as compared to those in the MDV-alone treatment. Therefore, it is suggested that the two designated doses of 25OHD<sub>3</sub> have the potential to be effectively combined with the MDV for subsequent administration by <i>in ovo</i> injection.
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