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Testing of parenteral pharmaceuticals and medical devices for pyrogens (fever-inducing substances) is critical to patient safety. The original rabbit pyrogen test has largely been replaced by different bacterial endotoxin tests based on Limulus amebocyte lysate (LAL), sourced from the blood equivalent of horseshoe crabs after comparative studies to the rabbit pyrogen test. Since 2004 a bacterial endotoxin test based on recombinant factor C (rFC), the endotoxin sensor protein inside of LAL, has been used as an animal-free alternative to LAL. Likewise, numerous studies compared LAL and rFC. Here we describe the history of pyrogen and bacterial endotoxin testing and summarize the evidence presented by those studies. We demonstrate that rFC and LAL are equivalent and comparable.

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 (

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 (

Although the African elephant (Loxodonta africana) is classified as endangered by the International Union for Conservation of Nature (IUCN), in some isolated habitats in southern Africa, contraception is of major interest due to local overpopulation. GnRH vaccination has been promoted as a non-invasive contraceptive measure for population management of overabundant wildlife. We tested the efficacy of this treatment for fertility control in elephant bulls.In total, 17 male African elephants that were treated with a GnRH vaccine were examined in two groups. In the prospective study group 1 (n = 11 bulls, ages: 8-36 years), semen quality, the testes, seminal vesicles, ampullae and prostate, which were all measured by means of transrectal ultrasound, and faecal androgen metabolite concentrations were monitored over a three-year period. Each bull in the prospective study received 5 ml of Improvac® (1000 μg GnRH conjugate) intramuscularly after the first examination, followed by a booster six weeks later and thereafter every 5-7 months. In a retrospective study group (group 2, n = 6, ages: 19-33 years), one examination was performed on bulls which had been treated with GnRH vaccine for 5-11 years.In all bulls of group 1, testicular and accessory sex gland sizes decreased significantly after the third vaccination. In six males examined prior to vaccination and again after more than five vaccinations, the testis size was reduced by 57.5%. Mean testicular height and length decreased from 13.3 ± 2.6 cm x 15.2 ± 2.8 cm at the beginning to 7.6 ± 2.1 cm x 10.2 ± 1.8 cm at the end of the study. Post pubertal bulls (>9 years, n = 6) examined prior to vaccination produced ejaculates with viable spermatozoa (volume: 8-175 ml, sperm concentration: 410-4000x106/ml, total motility: 0-90%), while after 5-8 injections, only 50% of these bulls produced ejaculates with a small number of immotile spermatozoa. The ejaculates of group 2 bulls (vaccinated >8 times) were devoid of spermatozoa. Faecal androgen metabolite concentrations measured in captive males decreased significantly after the fourth vaccination. None of the males entered musth during the treatment period.Our results showed a marked decrease in semen quality, testicle and secondary sex gland sizes following repeated GnRH vaccinations. After 2-4 years of continuous treatment every 5-7 months, the effects were similar to surgical castration.