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Recent loss of rhinoceros subspecies has renewed interest in using more advanced assisted reproductive technologies (ART) in rhinoceroses and elephants. Currently, only semen collection, semen preservation and artificial insemination (AI) have been used repeatedly with success in these species. Although ovum pick-up (OPU) and intra-cytoplasmic sperm injection (ICSI) have been reported recently in rhinoceroses, the techniques are not yet optimised. In contrast, multiple ART applications are routinely used in the horse. Since elephants and rhinoceroses share some reproductive features with equids, we postulate that procedures such as OPU, ICSI, in vitro fertilisation (IVF) and embryo transfer (ET), which are well established in the horse, may represent a basis to develop protocols for endangered pachyderms. In this review, we summarise current knowledge on reproductive physiology relevant to ART. We discuss the current state of ART in all three families and the requirements for the successful implementation of OPU, ICSI, IVF and ET in these species.

Sexual maturity and body condition are interconnected in many mammals. For non-domestic felids held in human care, the effect of body condition on male fertility has not been studied, although obesity is a recognized problem for many species such as the African lion (Panthera leo) under captive conditions.Here, we assessed body weight, body condition via body condition score (BCS), reproductive organ appearance and size by ultrasonography and semen parameters as a function of age in a large population of captive male lions housed in different facilities in South Africa. Of 59 individuals (age range 2.5–10 years), we rated 21 (36 %) of the males (≥ 4 years) as highly obese (BCS 8–9). Semen collection via urethral catheter was successful in 49 males (83.0 %). Sperm were found in 44 males (74.6 %) across all age groups. Sperm motility and detailed sperm morphology was assessed in 42 and 18 lions, respectively.As expected, body weight and reproductive organ size increased with age, but only the testis size increased in proportion to BCS. Although our data are unbalanced, as older animals (already after five years of age) were more often obese, they suggest an optimum age and body condition for potential fertility with adequate physical maturity in male lions. Physical maturity appeared to be reached between the ages of three and five years, which is later than sexual maturity in male lions.Lions that far exceeded the normal body weight range and had a BCS of 8 or 9 had lower semen quantity and quality. Therefore, male lions in captivity are in prime breeding condition when physical maturity is combined with a BCS < 8.Our data contribute towards interpretation of fertility assessments in managed breeding programs for lions. Ultrasonographic appearance of the reproductive organs, BCS and semen parameters can help to identify and select breeding males.

To develop successful ex-situ breeding programs for the conservation of threatened wildlife, an understanding of reproductive endocrinology in the species of interest is essential. Fecal hormone metabolite patterns are widely used in this regard, but limited information is available regarding how these reflect blood hormone profiles. This study aimed to longitudinally monitor and compare circulating progestagen (sP) and estrogen (sE) concentrations, as well as fecal progestagen (fPM) and estrogen (fEM) metabolite concentrations with observed reproductive behaviors of African lionesses. For 18 months, blood sampling (n = 309) from five captive lionesses took place 1–7 times per week during positive reinforcement training. In parallel, over a period of 9 months, fecal samples (n = 129) from four of these lionesses were searched for daily and collected when found. Daily behavioral monitoring of all females enabled classification of reproductive stage according to presence/absence of characteristic behavioral events (i.e., “allowing mount”, “copulation”, “flirting run”, “lordosis”, “purring”, and “rolling”). Competitive enzyme immunoassays were used for steroid quantification. In total, 11 an-ovulatory and 16 ovulatory cycles (7 pregnancies and 9 pseudo-pregnancies) were assessed. When compared, the pattern of fecal hormone metabolite concentrations matched reproductive behavior more reliably than circulating steroid concentrations. Both sP and fPM patterns were correlated (n = 51; r = 0.86) and enabled identification of luteal phases, which helped to distinguish between pregnant and non-pregnant females. In contrast, detection of estrus by measurement of sE and fEM was not accurate regardless of the matrix evaluated.

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 (

Background: Androgen-related aggressive behaviour and musth cause serious problems in captive African elephant bulls and often lead to human and animal injuries, and damage to property. Objectives: To review the work carried out with anti-gonadotropin-releasing hormone (GnRH) vaccines to control androgen-related behaviour and fertility in captive and free-ranging elephant bulls and the induction of anoestrus in elephant cows. Method: In the first study, an anti-GnRH vaccine from Pepscan was tested in six bulls (four captive and two free-ranging). Once the vaccine Improvac® became available, the effect on behaviour, the reproductive organs and semen quality was tested. Improvac® was also used to attempt induction of anoestrus in elephant cows. Results: The first study proved that aggressive behaviours are significantly associated with increased faecal androgen concentrations. Musth (n = 1) and aggressive behaviour (n = 2) were down regulated and correlated with a decline in faecal androgen concentrations. Aggression and musth could be controlled with Improvac® (600 µg), but were more consistent when the dose was increased to 1000 µg administered every five to six months. The same dose down regulated testicular function and bulls (n = 17) were rendered infertile within 12 months after commencement of treatment. Initial attempts to induce anoestrous with 600 µg in free-ranging elephant cows gave inconclusive results, but 1000 µg in captive cows delivered five-monthly was successful. Conclusion: The treatment of elephant bulls with Improvac® resulted in the successful down-regulation of androgen-related behaviour and sperm production in captive and wild elephant bulls of various ages (≤ 34 years). Preliminary studies to induce anoestrus in cows with Improvac® appear to be successful.

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.