The objective of the present study was planned to compare the response of donor Holstein cows to a GnRH-based (GnRH-SOV, n=9) and estradiol-based (E2-SOV, n=10) protocols. The super-stimulation protocols consisted of twice daily decreasing doses of porcine FSH with a total dose of 400 mg and the cows in both groups were sampled three times, before initiation of superovulation, at estrus and at embryo collection from donors. Circulating levels of FSH, LH, progesterone, estrogen and anti-Mullerian hormone (AMH), as well as response to superovulation were compared between the two groups. Results revealed that embryo yield and quality did not differ (P<0.05) between the two groups. When circulating concentrations of hormonal biomarkers were tested for possible correlations with embryo quality or quantity, progesterone was correlated with total embryo (r=0.71, P<0.01), total transferable embryo (r=0.84, P<0.01), second grade and third grade embryo (r=0.72 and 0.73, respectively; P<0.01) regardless of the time of sampling. Strong positive correlations were recorded between circulating levels of AMH prior to superovulation and embryo yield (total embryo, r=0.50; P<0.01), and quality (first grade embryo, r=0.78, P<0.01; second grade and third grade embryo, r=0.47 and 0.50, respectively with P<0.05). In conclusion, donor Holstein cows responded successfully to GnRH-SOV and E2-SOV protocols and circulating levels of progesterone and AMH could predict donor response to superovulation in Holstein cows.