Information regarding the physiology of African lions is scarce, mainly due to challenges associated with essential routine research procedures. The aim of this experiment was to test the possibility of training six captive lionesses by positive reinforcement conditioning (PRC) to voluntarily allow the collection of vaginal swabs and blood samples. This was done with the final goal of avoiding frequent anesthesia, and potential stressful management during research. All lionesses mastered basic clicker and targeting principles within two weeks. Routine sampling was possible after 20 weeks of training, enabling collection of about 750 vaginal swabs and 650 blood samples over the course of the study (18 months). These samples served to describe in detail the African lioness' ovarian cycle by combination of behavioral observations, longitudinal steroid hormone monitoring, and vaginal cytology. They also helped establish the ideal time for ovulation induction and artificial insemination of lionesses presenting natural estrus. The animals remained calm and cooperative during all sessions, and demonstrated curiosity in the training. PRC training of captive lionesses proved to be a suitable, minimally 1 invasive method for repeated collection of vaginal swabs and blood. Additionally, PRC may serve as behavioral enrichment for African lions in captive settings. Compared to chemical or physical restraining methods, this non-invasive management approach may reduce distress and physiological negative side effects, thus opening up new avenues for feline research.

Sexual selection involves mate choice and intrasexual competition (Darwin, 1871), an evolutionary mechanism that regulates reproductive success. Male-male competition in size dimorphic ungulates has been extensively studied (Andersson, 1994;Hirotani, 1989;Lent, 1965). Larger animals or individuals with larger weapons are more likely to win (Parker, 1974), hence have a greater chance to mate (Andersson, 1994). Among many mammals, male reproductive tactics involve tracking female endocrine state, with escalated aggression and mating occurring when females are most likely to conceive (Andersson, 1994;

Conservation management interventions for the critically endangered black rhinoceros (Diceros bicornis) require immobilization, which offer opportunities for semen collection and cryopreservation to establish genetic reservoirs. In free-ranging rhinoceroses, a combination of the potent opioid etorphine and the tranquilizer azaperone is routinely used for chemical immobilization but is associated with muscle rigidity and severe cardiopulmonary changes. Additionally, azaperone inhibits semen emission. Seven free-ranging, male, sexually mature black rhinoceroses were immobilized with an alternative protocol consisting of 4.5 mg etorphine, 5 mg medetomidine, 50 mg midazolam and 2,500 IU hyaluronidase delivered remotely by darting from a helicopter. During the immobilization, electro-ejaculation was performed with a portable electro-ejaculator, and a species-specific rectal probe. Animals were observed for muscle tremors. Longitudinal changes in respiratory rate, heart rate and peripheral oxyhemoglobin saturation, measured at 5 min intervals, were assessed using a general mixed model. Non-invasive oscillometric blood pressure and arterial blood gas variables were measured at first handling and before reversal and compared using the Wilcoxon rank sum test. All animals were successfully immobilized, showed no muscle tremors, presented with normal heart rates and lactate concentration (<5 mmol/L), recovered uneventfully, but experienced acidemia, hypoxemia and hypercapnia. Induction time and total time in recumbency were 4.2 ± 0.41 and 38.4 ± 6.9 min, respectively. Electro-stimulation commenced after 11.7 ± 3.98 min and completed after 24.3 ± 6.65 min. Semen-rich fractions were successfully collected from six animals. Our observations indicate that etorphine-medetomidine-midazolam provides a promising immobilization protocol for free-ranging black rhinoceroses, that allows for successful electro-ejaculation.

Disorders of sexual development (DSD) in wild mammals are rarely described. A male South African giraffe (Giraffa camelopardalis giraffa) was identified with bilateral cryptorchidism. The testes were intra-abdominal, smaller and less ovoid than in normal male giraffes. The right testis was situated more cranially than the left and connected to a longer deferent duct with normal ampullae. One distended vesicular gland filled with mucoid material was identified. A short penis, situated in the perineal area, was directed caudally and presented hypospadias. Histologically, testicular hypoplasia was present; the epididymis tubules contained no spermatozoa and the deferent duct and vesicular gland were inflamed. The blood testosterone concentration was 16.27 nmol/L and oestrone sulphate concentration was 0.03 ng/mL. The aetiology of the abnormalities is unknown.

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 (<5 mmol/L), experienced no muscle tremors and recovered uneventfully. Hypoxemia and hypertension persisted throughout the immobilization in all groups. Acidemia and hypercarbia were absent in Group 1, but present in the game-farmed animals. The lower etorphine dose in Group 3 resulted in significantly longer induction times, however, tachycardia was not observed. SpO2 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.