Efficacy and Safety of Native and Recombinant Zona Pellucida Immunocontraceptive Vaccines Formulated with Non-Freund's Adjuvants in Donkeys.

This study aimed to test zona pellucida (ZP) vaccines' immunocontraceptive efficacy and safety when formulated with non-Freund's adjuvant (6% Pet Gel A and 500 Μg Poly(I:C)). Twenty-four jennies were randomly assigned to three treatment groups: reZP (n = 7) received three doses of recombinant ZP vaccine; pZP (n = 9) received two doses of native porcine ZP; and Control group (n = 8) received two injections of placebo. Jennies were monitored weekly via transrectal ultrasonography and blood sampling for serum progesterone profiles and anti-pZP antibody titres. In addition, adverse effects were inspected after vaccination. Thirty-five days after the last treatment, jacks were introduced to each group and rotated every 28 days. Vaccination with both pZP and reZP was associated with ovarian shutdown in 44% (4/9) and 71% (4/7) of jennies, 118 ± 33 and 91 ± 20 days after vaccination, respectively (p > 0.05). Vaccination delayed the chances of a jenny becoming pregnant (p = 0.0005; Control, 78 ± 31 days; pZP, 218 ± 69 days; reZP, 244 ± 104 days). Anti-pZP antibody titres were elevated in all vaccinated jennies compared to Control jennies (p < 0.05). In addition, only mild local injection site reactions were observed in the jennies after treatment. In conclusion, ZP vaccines formulated with non-Freund's adjuvant effectively controlled reproduction in jennies with only minor localised side effects.

Effects of an anti-gonadoliberin releasing hormone vaccine on testicular, epididymal and spermatogenic development in the horse.

The effects of the GnRH vaccine Improvac® on testicular and epididymal morphometrics, histology and spermatogenesis were measured in 19 young (15-20 months) colts randomly assigned to one control (saline, castration at 57 days, n = 6) or either of two GnRH vaccine-treatment groups, T-57 (castration at 57 days, n = 7) or T-100 (castration at 100 days, n = 6), respectively. All were immunized on Day 0 with a single booster on Day 28. Excised testes and epididymides were weighed and processed for histology to measure tubule, epithelial and muscle dimensions, the ratio of interstitial tissue to seminiferous tubules and determine the stage of spermatogenesis. Testis volume, unchanged within controls, decreased in T-57 and T-100 groups by 50% and 70%, respectively. Treated colts' testes were significantly lighter than controls (64% relative difference); however, epididymal mass showed no significant differences between groups. Proportionally less seminiferous tubule relative to interstitial tissue was observed in both treatment groups (5%) versus controls (22%) with a mean tubule size 28% smaller than controls. Controls exhibited a high proportion of seminiferous tubules with advanced stages of spermatogenesis, whereas treated colts showed a high proportion of tubules in the early stages of spermatogenesis. In conclusion, immunization against GnRH in prepubertal colts was effective at reducing the development of their intra-scrotal reproductive organs and preventing normal spermatogenesis. GnRH vaccination of young colts effectively and consistently reduced testis mass, tubule size and relative proportion of seminiferous tubule tissue while retarding spermatogenesis. The epididymis showed changes with a smaller tubule diameter, lower epithelial height and thicker muscle layer recorded in treated compared to control colts.

Arterial Blood Gases and Cardiorespiratory Parameters in Etorphine-Medetomidine-Midazolam Immobilized Free-Ranging and Game-Farmed Southern White Rhinoceroses (Ceratotherium simum simum) Undergoing Electro-Ejaculation.

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.

Cardiopulmonary Parameters and Arterial Blood Gases During Etorphine-Medetomidine-Midazolam Immobilization in Free-Ranging Black Rhinoceroses (Diceros bicornis) Undergoing Electro-Ejaculation-A Preliminary Study.

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.

Social and Seasonal Factors Contribute to Shifts in Male African Elephant (Loxodonta africana) Foraging and Activity Patterns in Kruger National Park, South Africa.

African savannah elephants (Loxodonta africana) are well-known as ecosystem engineers with the ability to modify vegetation structure. The present study aimed to examine how male elephant foraging behaviour is affected across (a) season (wet versus dry); (b) time of day (before or after noon); (c) presence or absence of other elephants; and (d) reproductive state (musth versus no musth). Six radio-collared adult elephant bulls were observed twice per week from June 2007-June 2008 in Kruger National Park (KNP), South Africa. Using generalized linear mixed effect modeling, results indicate that elephant bulls graze more during the wet season and browse more during the dry season. To potentially offset the costs associated with thermoregulation during the heat of the day, KNP elephants spent more time foraging during the morning, and more time resting during the afternoon. Male elephants also foraged significantly less when they were associated with females compared to when they were alone or with other males. This is likely due to male-female associations formed mainly for reproductive purposes, thus impeding on male foraging behaviours. In contrast, the condition of musth, defined by the presence of related physical signs, had no significant effect on foraging behaviour.

Vaccination against GnRH as a prelude to surgical castration of horses.

BACKGROUND: Surgical castration is one of the most frequently performed surgeries in horses and is associated with various post-surgical complications ranging in severity. OBJECTIVES: We investigated the effects of administering an anti-GnRH vaccine to colts for reduction of testis size on the incidence of complications associated with their subsequent surgical castration. STUDY DESIGN: Randomised open controlled trial. METHODS: Nineteen colts were randomly assigned to one of the three groups. Treatment Groups V1-57 and V2-100 received two treatments of Improvac® (Zoetis, South Africa) before their castration in the field on either Day 57 or Day 100 following first treatment, respectively. Controls similarly received placebo treatments followed by castration on Day 57. Serial measurements of testicular dimensions and serum samples for measuring serum testosterone concentrations (STCs) and anti-GnRH antibody titres (ABTs) were obtained pre- and post-vaccination and on the day of castration. Clinical data recorded for 10 days determined post-surgical complications. RESULTS: All vaccinated colts showed a baseline STC concentration at castration with a strong ABT response. Mean testicular volume of Groups V1-57 and V2-100 reduced by 49.7% and 30.8%, respectively, on Day 57 and this further reduced in Group V2-100 by 63.9% at castration on Day 100. Testis size was significantly correlated with post-surgical preputial (P = 0.001) and scrotal (P = 0.025) swelling. MAIN LIMITATIONS: A study population of young light horse breed colts and a relatively small sample size limited this pilot study. CONCLUSIONS: Administering two doses of Improvac® in colts 28 days apart prior to castration effectively reduced their testicular volumes with associated significant post-surgical improvement in both the incidence and degree of local swelling. These results may inform mitigation of potential post-surgical complications associated with castration in the field.

Efficacy and safety of native and recombinant zona pellucida immunocontraceptive vaccines in donkeys.

Feral and semi-feral donkeys are recognised as a problem in some world regions. The main problem associated with uncontrolled donkey populations is habitat degradation and competition for feed resources, especially in arid climes. Controlling population numbers would reduce the impact of donkeys and other species. While removal by various means is effective, it has been shown to stimulate reproductive rate. Probably the most effective and humane solution is reducing reproduction using minimally invasive methods including immunocontraception. This study tested the immunocontraceptive efficacy and safety of zona pellucida (ZP) vaccines, both recombinant (reZP; three treatments) and native porcine (pZP; two treatments) vaccines formulated with Freund's modified complete (primary) and Freund's incomplete (boosters) adjuvants in donkey jennies. Control jennies received adjuvants only (two treatments). Twenty-five non-pregnant jennies were randomly assigned to reZP (n = 9), pZP (n = 8) or control (n = 8) groups. Weekly monitoring of the reproductive tract and ovaries via transrectal palpation and ultrasound and inspection of injection sites was conducted and anti-pZP antibody titers were measured. Five weeks after last treatment, one donkey jack was introduced to each group and rotated every 21 days. By 232 days after last treatment the number pregnant and median days to pregnancy was 2/9 and 214 (reZP group), 1/8 and 196 (pZP group) and 8/8 and 77 (control group). Median time to ovarian shut-down was 77 (9/9) and 56 (7/8) days for reZP and pZP groups, respectively. This was observed in association with a distinct reduction in mean uterine diameter. The antibody response was equally good for both ZP-treated groups. Incorporation of Freund's adjuvants initially produced a high incidence of side effects from local swelling and intermittent lameness followed weeks later by sterile abscesses (reZP, 9/9; pZP, 7/8; control, 3/8). Both ZP vaccines effectively controlled reproduction in jennies, albeit with a high incidence of adjuvant-associated side effects.

Researching immunocontraceptive vaccines with mares (Equus caballus) as both a target and model for African elephant (Loxodonta africana) cows: A review.

A sequence of studies is reviewed that reported the domestic horse (Equus caballus) mare as an appropriate and accessible research platform for recording clinical and laboratory data post-immunisation with anti- GnRH and -zona pellucida (ZP) immunocontraceptive vaccines. Experience with a native porcine ZP (pZP) vaccine in African elephant (Loxodonta africana) cows highlighted needs for improving vaccine formulations and more clearly defining associated ovarian effects and safety profiles. Initially, the efficacy, reversibility and safety of the GnRH vaccine Improvac® in mares was demonstrated using reproductive tract ultrasonography and concurrently measuring serum antibody titres and progesterone concentrations. Results informed the study design and minimally invasive monitoring of post-treatment ovarian steroid responses of this vaccine in free-ranging African elephant cows. A subsequent sequence of studies reported reversible contraceptive and immunological efficacy in pony mares immunised with pZP formulated with Freund's adjuvants. By comparison, mares treated with a recombinant ZP3 and ZP4 (reZP) vaccine showed disappointing responses. Unexpectedly, most pZP-treated mares showed ovarian inactivity. In attempting to understand this response, results showed the involvement of cytotoxic (CD8+) T-cells negatively correlated to serum ovarian steroid and anti-Müllerian hormone (AMH) levels. Of concern was the prevalence of injection-site lesions ascribable to Freund's adjuvants. Following this, mares treated with both pZP and a novel reZP vaccine formulated with non-Freund's adjuvants showed comparable immunological responses and ovarian inactivity, notably without adverse treatment reactions. In addition, measuring AMH showed promise for monitoring ovarian function in anti-ZP-treated animals.

Serum antibody immunoreactivity and safety of native porcine and recombinant zona pellucida vaccines formulated with a non-Freund's adjuvant in horses.

Commercial and regulatory limitations associated with native porcine zona pellucida (pZP) vaccines formulated with Freund's adjuvants may be overcome by developing effective recombinant ZP vaccines (reZP) and identifying alternative adjuvant formulations. A two-part preparatory study used 15 geldings and identified potentially effective alternative adjuvant formulations based on anti-pZP antibody response following treatment with pZP formulated with Addavax (AddaVax ™, Invivogen), Quil A (Quil-A® Adjuvant, Invivogen), Quil A and Poly (I:C) (HMW VacciGrade™, Invivogen), Pet Gel A (Montanide™ Pet Gel A, Seppic) and Pet Gel A and Poly (I:C). Injection site reactions, rectal temperature and respiratory and heart rates were also monitored for three days post-treatment. Suitable anti-pZP antibody titres were seen in response to Pet Gel A and Pet Gel A and Poly (I:C). Subsequently in 31 mares, following administration of pZP, reZP and a combination of pZP and reZP proteins prepared in Pet Gel A and Poly (I:C), both serum anti-pZP and -reZP antibody responses were monitored. In addition, safety was assessed for up to seven days post-treatment by inspection and palpation of gluteal intramuscular injection sites and measurement of rectal temperature. The measured antibody titres in all treatment groups differed significantly to an adjuvant control group (P < 0.001). Temporal changes in both anti-pZP and -reZP antibody titres in all ZP treatment groups were similar to patterns reported previously in various species vaccinated with pZP formulated with Freund's adjuvants. There were no differences in anti-pZP antibody titres between the pZP and reZP treated groups (P > 0.05). Side effects were mild and transient in nature. This represents the first application of a reZP vaccine formulated with non-Freund's adjuvants evoking a similar antibody titre response to native pZP vaccination in mares.

Ovarian function following immunocontraceptive vaccination of mares using native porcine and recombinant zona pellucida vaccines formulated with a non-Freund's adjuvant and anti-GnRH vaccines.

An important determinant in the selection of any contraceptive agent is the impact on ovarian function, both in the short and longer term. In this study, ovarian activity was monitored in mares immunised with one of the following vaccine formulations; native porcine zona pellucida (pZP), recombinant zona pellucida proteins ZP3 and ZP4 (reZP), pZP and reZP combined or a commercially available anti-GnRH vaccine. The ZP antigens were prepared in an adjuvant formulation consisting of 6% polymeric adjuvant (Montanide™ PetGel A, Seppic, France) and 500 µg polyinosinic-polycytidylic acid - TLR3-agonist (Poly(I:C) HMW VacciGrade™, Invivogen, USA). A vehicle-only control group was administered the adjuvant formulation without antigen. Ovarian activity was monitored using clinical observations (transrectal palpation and ultrasonography of the reproductive tract) in addition to blood sampling for serum progesterone and anti-Müllerian hormone (AMH) concentrations while employing a low sampling frequency. Treatments and measurements were initiated in December (southern hemisphere summer) and subsequent data collection was performed in January, February, March and May. Both reZP and anti-GnRH vaccination were associated with clinically evident ovarian suppression in the short term. Ovarian activity in mares administered a reZP or anti-GnRH vaccine was significantly different to adjuvant control and pZP treated mares. Serum AMH concentrations were different between pZP and anti-GnRH treated mares 3.5 months after the final vaccination. Serum AMH concentrations were significantly correlated with mare age, serum progesterone and ovarian volume.

Effects of GnRH vaccination in wild and captive African Elephant bulls (Loxodonta africana) on reproductive organs and semen quality.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Ovarian dysfunction associated with zona pellucida-based immunocontraceptive vaccines.

Despite more than 40 years of research into zona pellucida (ZP)-based vaccines, relatively little is known about their mechanism of action. Early research demonstrated precipitation of ZP glycoproteins by antiovarian antiserum, rendering oocytes resistant to sperm binding in vitro. Subsequent work showed significantly decreased fertilization rates following passive immunization, sparking interest in anti-ZP immunocontraception for human and animal use. The primary mechanism of action of ZP vaccines is generally considered to be an antibody-mediated interference with sperm-oocyte binding and/or fertilization. However, this mechanism of action excludes the potential for ovarian dysfunction associated with anti-ZP treatment in some species. A review of relevant literature in pertinent model, domestic and wildlife species reveals a variety of previous and current hypotheses for ovarian effects following ZP-based immunization. Ovarian dysfunction has been suggested to be a species-specific response. In addition, cytotoxic T-lymphocytes and the use of Freund's adjuvants have been suggested to play a role. Finally, the type and extent of glycosylation of ZP antigens have been proposed to influence ovarian effects. The validity of these hypotheses is re-examined in the light of current knowledge. Further investigation of ovarian function in species believed to be resistant to the ovarian effects of anti-ZP vaccines is warranted. To this end, anti-Müllerian hormone may provide a novel tool for the assessment of ovarian function during ZP-based immunocontraception, particularly in wildlife species not amenable to frequent clinical examination.

Lack of spatial and behavioral responses to immunocontraception application in African elephants (Loxodonta africana).

Opinions are divided as to whether human intervention to control elephant (Loxodonta africana) population growth is desirable, partly because of elephant welfare concerns. Female contraception through immunization with porcine zona pellucida (PZP) proteins is viable. The effects of sustained use and application of the PZP vaccine on elephant behavioral and spatial responses were examined by evaluating herd ranging, fission-fusion dynamics, association patterns, and reproductive and sexual behaviors. Minimal change was anticipated as a result of long calf dependence on and association with cows, a reduced but not indefinite 0% growth rate and the known mechanism of action of PZP vaccines, and minimal expected change in resource requirements necessitating behavioral or spatial use adaptations. Although behavioral effects identified in previous hormonal contraceptive trials were evident, it was demonstrated that immunocontraception caused no prolonged behavioral, social, or spatial changes over the 11-yr study period. Individually identified elephants were monitored from 1999 to 2011. Minimal, short-term social disruption, with temporary changes to the herds' core ranges, was observed during the annual treatment events, particularly in the first three treatment years, when vaccinations were conducted exclusively from the ground. Thereafter, when vaccinations were conducted aerially, minor disruptions were confined to the morning of administration only. Despite sustained treatments resulting in demographic changes of fewer calves being born, treatments did not alter spatial range use, and no adverse interherd-intraherd relations were observed. Similarly, resource requirements did not change as calving still occurred, although in fewer numbers. It was concluded that PZP immunocontraception has no detectable behavioral or social consequences in elephants over the course of 11 yr, providing a convincing argument for the use of sustained immunocontraception in the medium to long term as an important tool for elephant management. Behavioral consequences of alternative management approaches should all receive similar scrutiny to enable managers to make informed decisions when weighing management interventions.

Non-invasive assessment of the reproductive cycle in free-ranging female African elephants (Loxodonta africana) treated with a gonadotropin-releasing hormone (GnRH) vaccine for inducing anoestrus.

BACKGROUND: In southern Africa, various options to manage elephant populations are being considered. Immunocontraception is considered to be the most ethically acceptable and logistically feasible method for control of smaller and confined populations. In this regard, the use of gonadotropin-releasing hormone (GnRH) vaccine has not been investigated in female elephants, although it has been reported to be safe and effective in several domestic and wildlife species. The aims of this study were to monitor the oestrous cycles of free-ranging African elephant cows using faecal progestagen metabolites and to evaluate the efficacy of a GnRH vaccine to induce anoestrus in treated cows. METHODS: Between May 2009-June 2010, luteal activity of 12 elephant cows was monitored non-invasively using an enzyme immunoassay detecting faecal 5alpha-reduced pregnanes (faecal progestagen metabolites, FPM) on a private game reserve in South Africa. No bulls of breeding age were present on the reserve prior to and for the duration of the study. After a 3-month control period, 8 randomly-selected females were treated twice with 600 micrograms of GnRH vaccine (Improvac®, Pfizer Animal Health, Sandton, South Africa) 5-7 weeks apart. Four of these females had been treated previously with the porcine zona pellucida (pZP) vaccine for four years (2004-2007). RESULTS: All 12 monitored females (8 treated and 4 controls) showed signs of luteal activity as evidenced by FPM concentrations exceeding individual baseline values more than once. A total of 16 oestrous cycles could be identified in 8 cows with four of these within the 13 to 17 weeks range previously reported for captive African elephants. According to the FPM concentrations the GnRH vaccine was unable to induce anoestrus in the treated cows. Overall FPM levels in samples collected during the wet season (mean 4.03 micrograms/gram dry faeces) were significantly higher (P<0.002) than the dry season (mean 2.59 micrograms/gram dry faeces). CONCLUSIONS: The GnRH vaccination protocol failed to induce anoestrus in the treated female elephants. These results indicate that irregular oestrous cycles occur amongst free-ranging elephants and are not restricted to elephants in captivity. The relationship between ecological conditions and endocrine activity were confirmed. Free-ranging female elephants were observed to not cycle continuously throughout the year in the absence of adult bulls.

Seasonal changes in steroid hormone profiles, body weight, semen quality, and the reproductive tract in captive African wild dogs (Lycaon pictus) in South Africa.

Characterization of reproductive seasonality in the African wild dog (Lycaon pictus) could assist reintroduction programs. Male wild dogs (n=14) were assessed quarterly (January, mid-summer; April, late summer; August, late winter; November, early summer) for serum testosterone, body weight, testicular and prostatic volume, preputial gland measurement, and ejaculate characteristics. Bi-monthly fecal samples were collected from male (n=11) and female (n=4) wild dogs for analysis of fecal androgens and progestagens. Fecal androgens were higher in early summer (246.4 ± 14.5 ng/g) than in early winter (218.6 ± 13.4 ng/g). Serum testosterone was higher in mid-summer (1.4 ± 0.3 ng/ml) than in late winter (0.7 ± 0.1 ng/ml). Number of spermatozoa per ejaculate was greatest in late summer (301.4 ± 39.3 × 10(6)). Other semen parameters peaked in mid-summer (pH: 7.4; progressive motility: 85.0 ± 0.1%; live spermatozoa: 81.0 ± 16%; normal morphology: 71.5 ± 8.2%). Total testicular and prostatic volume were greater during summer (testicular: 36.7 ± 4.2 cm(3); prostatic: 12.0 ± 1.9 cm(3)) than winter (testicular: 25.2 ± 1.9 cm(3); prostatic: 5.8 ± 0.8 cm(3)). Preputial pendulance also was greater in summer (7.1 ± 0.5 cm; n=9) than winter (5.9 ± 0.2 cm). Baseline fecal progestagen metabolites were 6.2 ± 2.5 µg/g and peak fecal progestagen metabolites were 14.7 ± 2.8 µg/g. Copulations resulting in pregnancies (n=2) occurred in late summer and gestation was 71 days. Female wild dogs were seasonally monoestrous with mating in summer and winter. In conclusion, wild dogs are reproductively seasonal with improvement in male reproductive variables during summer and a bi-phasic seasonal pattern to female receptivity.

Validation of noninvasive monitoring of adrenocortical endocrine activity in ground-feeding aardwolves (Proteles cristata): exemplifying the influence of consumption of inorganic material for fecal steroid analysis.

Biologically inert material in feces may confound interpretations of noninvasive fecal endocrine data, because it may induce variance related to differences in foraging behavior rather than to differences in endocrine activity. We evaluated two different enzyme immunoassays (EIAs) for the noninvasive evaluation of adrenocortical activity in ground-feeding aardwolves (Proteles cristata) and tested the influence of soil content in aardwolf feces on the interpretation of fecal glucocorticoid metabolite data. Using adrenocorticotropic hormone (ACTH) challenges for validation, we successfully identified a cortisol EIA suitable for assessing adrenocortical activity in aardwolves. An alternatively tested 11-oxoetiocholanolone EIA failed to detect a biologically relevant signal after ACTH administration. Although the proportion of inorganic content in aardwolf feces did not alter qualitative conclusions from the endocrine data, the data related to mass of organic content had a larger amount of variance attributed to relevant biological contrasts and a lower amount of variance attributed to individual variation, compared with data related to total dry mass of extracted material. Compared with data expressed as dry mass of extracted material, data expressed as mass of organic content may provide a more refined and statistically powerful measure of endocrine activity in species that ingest large amounts of indigestible material.

Endocrine correlates of musth and the impact of ecological and social factors in free-ranging African elephants (Loxodonta africana).

Sexual activity in mature male African elephants is predominantly associated with the occurrence of musth, a state or condition which refers to a set of physical, physiological and behavioral characteristics, including an elevation in androgen levels. Although musth appears to be energetically costly, the degree to which it is associated with changes in adrenal endocrine function (e.g., glucocorticoid output) is still unclear. To investigate the possible effect of musth on adrenocortical function, and the impact of socioecological changes on androgen and glucocorticoid levels, six adult African elephant bulls were followed for 13months in the Kruger National Park, South Africa, and observations and fecal sample collection for endocrine monitoring was carried out about twice weekly. Our data showed that the occurrence of musth was associated with reduced glucocorticoid output, suggesting that musth does not represent a physiological stress mediated by the hypothalamic-pituitary-adrenal axis. This confirms previous findings in captive-housed animals, providing evidence for a suppressive effect of the musth condition on adrenocortical activity. Furthermore, a seasonal effect on androgen and glucocorticoid levels was found, which appears to vary depending on the reproductive status of the animal. The results also indicate a relationship between the presence or absence of social partners and changes in testicular and adrenal endocrine activity. Finally, the data confirm previous findings in captive-housed elephants, that an elevation in androgen concentrations usually occurs before the onset of physical musth signs, and therefore support the idea that the change in androgen levels represents the initial stimulus for the musth condition.

Molecular detection of Babesia rossi and Hepatozoon sp. in African wild dogs (Lycaon pictus) in South Africa.

Blood specimens from wild dogs (n=301) were obtained from De Wildt Cheetah and Wildlife Centre (Pretoria) and five game reserves (4 in the North-West Province and 1 in Limpopo Province), South Africa. Specimens were screened for Babesia, Theileria, Hepatozoon and Ehrlichia/Anaplasma species using PCR and Reverse Line Blot (RLB) assays. Positive results were obtained in 18 (6%) wild dogs. Sixteen specimens were found positive for Babesia rossi and two dogs were Hepatozoon sp. positive. It appears that these tick-borne pathogens are not widely distributed in wild dog populations.

Detection of feline coronavirus infection in southern African nondomestic felids.

Feline coronavirus (FCoV) infects members of the Felidae family with results ranging from seroconversion with no disease to fatal feline infectious peritonitis (FIP). Infection of non-domestic felids with FCoV is of concern, particularly in endangered populations such as cheetahs (Acinonyx jubatus). In this investigation, we tested 342 animals in the Republic of South Africa and Namibia, including 140 animals from wild populations, for evidence of FCoV infection by serology and/or reverse transcription/nested polymerase chain reaction (RT/nPCR) on feces from 1999 through 2001. Past or current infection was evaluated. Of these, 195 animals had evidence of infection and included 41 animals from wild populations. Serology (indirect immunofluorescence) did not always correlate with viral RNA detection, as seronegative animals were occasionally virus-positive, while many seropositive animals were not shedding virus. Serology indicated the infecting virus was most closely related to type I FCoV. Antibody levels in the majority of animals were low, even in those actively infected. Ten of 48 animals tested at more than one time point by RT/nPCR were shedding virus at multiple time points possibly indicating persistent infection. Infection in free-ranging animals was also notable, as over a quarter of the free-ranging animals tested had evidence of current or previous FCoV infection. Testing by serology and RT/nPCR is recommended for screening for FCoV infection.