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With the rapid loss of individuals in the wild, semen cryopreservation has gained importance to safeguard the genetic diversity of white rhinoceroses (Ceratotherium simum). For semen collection via electro-ejaculation, immobilization of free-ranging individuals requires the potent opioid etorphine, which is routinely combined with azaperone, but causes hypoxemia, hypercarbia, acidemia, muscle rigidity, tachycardia, and systemic hypertension. In this study, the suitability of two alternative immobilization protocols including etorphine, medetomidine, and midazolam at different doses (high vs. low etorphine) was evaluated in adult white rhinoceros bulls in two different management systems (free-ranging vs. game-farmed) and undergoing electro-ejaculation. Fourteen free-ranging (Group 1) and 28 game-farmed rhinoceroses (Group 2) were immobilized with ≈2.5 μg/kg etorphine (high dose), ≈2.5 μg/kg medetomidine, ≈25 μg/kg midazolam and 1,500–1,700 IU hyaluronidase and received ≈2.5 μg/kg of butorphanol intravenously at first handling. Twenty game-farmed animals (Group 3) received ≈1 μg/kg etorphine (low dose), ≈5 μg/kg medetomidine, ≈25 μg/kg midazolam and 1,700 IU hyaluronidase. Respiratory rate, heart rate and peripheral hemoglobin oxygen saturation (SpO2) were measured at 5-min intervals; non-invasive oscillometric blood pressures and arterial blood gases at first handling and before reversal of the immobilization; serum clinical chemistry analytes and hematocrit at first handling. Generalized mixed models (fixed factors: group, time, recumbency; random factor: individual rhinoceros) were applied to compare longitudinal changes between free-ranging and game-farmed rhinoceroses immobilized with the higher etorphine dose (Groups 1 and 2), and between the two protocols tested in the game-farmed rhinoceroses (Groups 2 and 3). All animals were successfully immobilized, presented with normal lactate concentrations (2 was higher for sternal vs. lateral recumbency. Semen-rich fractions were recovered following electro-stimulation in 46 out of the 62 animals. Our findings suggest that etorphine-medetomidine-midazolam provides effective immobilization with fewer side effects compared to previous reports in white rhinoceroses and is suitable for successful electro-ejaculation.