Comparative reproduction of the female horse, elephant and rhinoceros: implications for advancing Assisted Reproductive Technologies (ART).

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 in rhinoceroses, the techniques are not yet optimised. In contrast, multiple ART applications are routinely used in the horse. Since elephant 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 summarize 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.

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.

Construction of a full mouth speculum facilitating oral examinations, bronchoscopy and gastric tubing in elephants.

OBJECTIVE: Here we tested the application of a full mouth speculum to sedated elephants in human care to gain access to the oral cavity, the trachea (bronchi) and esophagus (stomach) and therefore improve diagnostic and therapeutic options in elephant medicine. The construction of this oral speculum for elephants and the procedure are described. MATERIAL AND METHODS: The oral speculum is a steel construction consisting of 2 bite plates of 0.8 × 60.0 × 8.0 cm attached between 2 threaded guiding poles (40 cm). Through crank handles, the metal plates are dispersed once placed between the elephant's jaws in front of the molars. The oral speculum was applied in 26 elephants (6,16 Asian elephants, and 1,3 African elephants) during standing sedation. RESULTS: All sedated elephants tolerated the positioning of the mouth opener and subsequent manipulations well. The mouth opener was applied for the following procedures: inspection of the oral cavity (n = 2), placing a stomach tube (n = 16), and/or performing endoscopic examinations such as bronchoscopy (n = 20) and/or gastroscopy (n = 8). CONCLUSION: This method provides a new possibility to open the jaws to gain access to the molars, larynx and pharynx in captive elephants without full immobilization. Valuable samples for diagnostics may be obtained or animals medicated via stomach tube with this application. CLINICAL RELEVANCE: The mouth opener provides veterinarains with a new option to perform necessary diagnostic and therapeutic procedures around the oral cavity, airways and stomach in captive elephants during standing sedation with no need for a full anaesthesia.

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.

TREATMENT OF MYCOBACTERIOSIS CAUSED BY MYCOBACTERIUM AVIUM SSP. HOMINISSUIS IN A GROUP OF CAPTIVE LOWLAND TAPIRS (TAPIRUS TERRESTRIS).

Tapirs are a taxonomic group with a high susceptibility to mycobacterial diseases. However, successful therapy has only been documented sporadically. Here treatment of mycobacteriosis diagnosed in three, one male and two female, lowland tapirs (Tapirus terrestris) in a zoo in Germany is reported. Two of the animals showed chronic mild respiratory signs, and conventional therapy did not improve the condition. Culture of broncho-alveolar lavage (BAL) samples was positive for Mycobacterium avium ssp. hominissuis. Upon airway endoscopy, bronchial edema and increased mucus production were visible. Initially, all three infected tapirs received oral antimycobacterial therapy consisting of 5 mg/kg body weight isoniazid, 10 mg/kg rifampicin, and 10 mg/kg clarithromycin q24h. Based on therapeutic drug level monitoring, the doses of rifampicin were adjusted to 12 and 15 mg/kg in the females and the male, respectively. The treatment with all three drugs was continued for 11 mon. Six months into treatment, the clinical condition resolved, and repeated BAL samples of all three tapirs tested negative for mycobacteria by culture. Here the approach for a treatment protocol with minimal side effects suitable to control infections with nontuberculous mycobacteria in lowland tapirs is reported.

Clinical outcome and diagnostic methods of atypical mycobacteriosis due to Mycobacterium avium ssp. hominissuis in a group of captive lowland tapirs (Tapirus terrestris).

Tapirs seem particularly susceptible to mycobacterial infections, especially to tuberculosis caused by M. tuberculosis or M. bovis. In this case series, we report an infection with the non-tuberculous mycobacteria (NTM) species M. avium ssp. hominissuis (MAH) in a group of four (2.2) captive lowland tapirs (Tapirus terrestris). Two female tapirs showed mild respiratory signs such as coughing and mucous sputum production for several years, one juvenile male tapir had to be euthanized due to severe dyspnoea, and the adult male only showed mild respiratory signs in 2010. Post-mortem histopathology of the euthanized animal revealed a chronic bronchopneumonia, and MAH was detected via culture. Subsequently, the three remaining tapirs were tested further: serologically, the tapirs had high antibody titres against M. avium, but they showed no reaction in the comparative skin test (TST). At several time points, the animals were tested for the presence of mycobacteria in different sample matrices including sputum samples, pooled faecal samples as well as swabs from the tapir enclosure to identify potential environmental niches of the pathogen. Moreover, animals were directly sampled using nasal swabs, endoscopic broncho-alveolar (BAL) and gastric lavages. MAH was detected by culture in the sputum samples, in the BAL of the breeding pair, as well as in the swimming pool water and walls, and in swabs taken from the tapir's sleeping beds. We conclude that the TST is not a useful diagnostic tool to detect MAC infections in tapirs, whereas antibody ELISA and culture from BAL appear more sensitive.

Managed wildlife breeding-an undervalued conservation tool?

Knowledge of and the technologies and resources applied to the ex situ care for wildlife have improved greatly in recent years. This has resulted in numerous successes bringing back populations from the brink of extinction by the reintroduction or restoration of animals from conservation breeding programmes. Controlled breeding of wildlife by humans is discussed controversially in society and in scientific circles and it faces a number of significant challenges. When natural breeding fails, Assisted Reproduction Technologies (ART) have been postulated to increase reproductive output and maintain genetic diversity. Furthermore, technical advances have improved the potential for successful collection and cryopreservation of gametes and embryos in many wildlife species. With the aim of creating a better understanding of why ex situ and in situ conservation of threatened species must complement each other, and under which circumstances ART provide additional tools in the rescue of a threatened population, we elucidate the current situation here by using as examples three different megavertebrate families: elephantidae, rhinoceridae and giraffidae. These mammal families consist of charismatic species, and most of their members are currently facing dramatic declines in population numbers. On the basis of these and other examples, we highlight the importance of captive zoo and other managed wildlife populations for species survival in a human dominated world. Without the possibility to study reproductive physiology in trained or habituated captive individuals, major advances made in wildlife ART during the past 20 years would not have been possible. This paper reviews the benefits and future challenges of large mammal conservation breeding and examines the role of assisted reproduction in such efforts.

Reproductive cycle stage assessment using vaginal cytology evaluation in African lions (Panthera leo).

Vaginal cytology evaluation is an economic, non-invasive technique for indirect monitoring of fluctuations in estrogen concentrations, and thus progression of the estrous cycle. This technique is widely used in domestic dogs for determining timing of artificial insemination. There, however, are only a few reports on the vaginal cytology of non-domestic felids, including lions. This study was conducted, therefore, to describe the vaginal epithelial changes throughout the reproductive cycle of African lions, and to investigate the efficacy of vaginal cytology assessments for predicting reproductive stages. During a 12-month period, reproductive behavioral data and vaginal swabs were collected daily from five lionesses. In total, 541 vaginal smears were evaluated for the proportion of mucosal epithelial cells, neutrophils, bacterial cells, and amount of mucous, cellular debris. One single swab with a large proportion of superficial cells, absence of neutrophils, large number of bacteria, without cellular debris was sufficient for detecting lionesses in estrus. Likewise, one cytology sample with a large proportion of parabasal and intermediate cells, few neutrophils, few bacteria, and large amount of mucous, cellular debris enabled detection of females in advanced diestrus or gestation. To distinguish lionesses in early diestrus from those in an inter-estrous period, at least two consecutive swabs were necessary for satisfactory classification. Overall, evaluation of vaginal cytology samples was an effective technique for differentiation among different stages of the reproductive cycle, confirmation of estrus, and pregnancy diagnosis in lionesses. This technique, therefore, has the potential for application in classifying different stages of the reproductive cycle in other feline species.

Positive Reinforcement Conditioning as a Tool for Frequent Minimally Invasive Blood and Vaginal Swab Sampling in African Lions (Panthera Leo).

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 potentially stressful management during reproduction research. All lionesses mastered basic clicker and targeting principles within 2 weeks. Routine sampling was possible after 20 weeks of training, enabling the collection of about 750 vaginal swabs and 650 blood samples over 18 months. 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 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 noninvasive management approach may reduce distress and physiological negative side effects, thus opening up new avenues for feline research.

Non-surgical artificial insemination using a GnRH analogue for ovulation induction during natural oestrus in African lions (Panthera leo).

Despite postulated potential for wildlife conservation, success of assisted reproduction techniques (ART) in ex-situ feline breeding remains <25%. The aim of this project was to develop a simplified, non-surgical artificial insemination (AI) protocol for African lions (Panthera leo), using an exogenous GnRH analogue to induce ovulation in females presenting natural oestrus, and minimizing manipulation of the animals. Four protocols were tested in five trained lionesses (3.5-8 years), for a total of 14 inseminations (2-4 per lioness). These protocols differed in the time lapse between GnRH injection and insemination, on days 4, 5, or 6 from onset of natural oestrus, determined by daily behavioural observation and vaginal cytology. Semen was collected from 8 different males by urethral catheterization and electro-ejaculation, during full anaesthesia. Females were immediately immobilized for AI after semen collection. After transrectal ultrasound examination of the reproductive tract, insemination was performed either intravaginal or transcervical using a commercial dog urinary catheter (2.0 × 500 mm, Buster®, Krusse, South Africa) with a metal stylet. A single intramuscular dose of exogenous GnRH (20 µg burserelin-acetate, Receptal®, MSD, Intervet, South Africa) administered 30 or 48 h before AI or during the AI procedure induced ovulation successfully, as all females entered either a non-pregnant luteal phase of 59.6 ±â€¯0.95 days (n = 10) or a pregnant luteal phase of 111.7 ±â€¯0.33 days (n = 3). However, the timespan between GnRH injection and end of behavioural and/or cytological oestrus differed widely (range: 0-120 h). The final pregnancy success rate was 33.3%.

Detection of Simonsiella spp. in the Vagina of Lions and Leopard in Oestrus.

Reports of the vaginal flora of wild cats such as lions or leopards are scarce. The micro-organisms most commonly found in the vagina of clinically healthy cats are aerobic bacteria such as coagulase-negative Staphylococcus, Streptococcus canis, and Escherichia coli. Simonsiella spp are large Gram-negative bacteria belonging to the Neisseriaceae family, typically found in the oral cavity and upper respiratory tract of many species. To date, there are no reports of the detection of Simonsiella spp in the vaginal flora of any felid. For a period of six months, daily behaviour monitoring was performed on six captive lionesses at a South African conservation centre, in parallel with the collection of vaginal swabs and interpretation of the resultant vaginal cytologies every other day. Oestrus was identified by typical female reproductive behaviours, as well as by enlarged and separated vulvar lips, and a predominant proportion of superficial cornified cells, clearing of the background, and high bacterial presence in the vaginal smear. Simonsiella spp were identified by their characteristic morphology in 58% (60 of 103) of the vaginal samples collected during oestrus. They were also found in oral swabs of three out of three lions tested. Additionally, Simonsiella spp were opportunistically found in a vaginal smear from a zoo housed female Sri Lankan leopard in oestrus, during a routine reproduction assessment. The finding of Simonsiella spp may be more common than previously suspected, transitory, and without detectable clinical relevance. A connection between occurrence of these bacteria and oestrus was apparent.

Prolonged luteal lifespan and pseudopregnancy in Asian elephants (Elephas maximus).

Pseudopregnancy is a physiological occurrence in mammals which have copulation induced ovulation, but is rarely described in spontaneous ovulating species. In this study, three cases of prolonged luteal lifespan are reported in non-pregnant Asian elephants (Elephas maximus). Case 1 was a 25-year-old female that had produced three calves previously; Case 2 was a nulliparous and 32-year-old at the start of the pseudopregnancy episode; and Case 3 occurred in a 49-year-old nulliparous elephant. Serum progesterone metabolite concentrations remained elevated for 10 months in Case 1. Urinary progestagens were high for >16 months in Case 2 and for five months in Case 3. In Case 1, multiple persistent corpora lutea were visualized monthly by ultrasonography. In all three cases, uterine leiomyoma were present and progestagen concentrations decreased spontaneously. In Case 1, the elephant became pregnant 3 years later, whilst with Case 2, the female resumed estrous cycling normally, and for the Case 3 female, there was continuation with another prolonged luteal phase before ovarian function was purposely suppressed. These examples indicate that persistently elevated progestagen concentrations may not always be indicative of pregnancy in elephants. The reasons for prolonged luteal lifespan are not understood, although serum prolactin concentrations quantified in the Case 1 female were elevated compared to values from previous reports and two other herd mates. Furthermore, all three elephants had varying degrees of uterine pathologies. It is believed that the resulting damage to the endometrium may have led to a reaction similar to implantation, which includes prolactin secretion. Prolactin may exert luteotropic properties and is thought to initiate luteal rescue during pregnancy in elephants.

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.

Unusual Outbreak of Fatal Clostridiosis in a Group of Captive Brown Pelicans ( Pelecanus occidentalis).

Fatal clostridial infections and clostridial toxicoses are common in birds. Most fatalities are associated with toxin production and progress rapidly, often within 24 hours of infection. We describe an unusual and protracted course of disease in 6 captive brown pelicans ( Pelecanus occidentalis), which was believed to result from toxicosis by toxovar A produced by a mixed infection with Clostridium sordellii and Clostridium perfringens. Although the first death in the group occurred 3 days after signs of illness were documented, the remaining birds died over a 38-day period despite aggressive antibiotic and supportive therapy. Although the birds presented with classic signs of botulism, Clostridium botulinum was not identified in any tissues or environmental samples. Postmortem findings in all pelicans included extensive subacute myonecrosis, enteritis, and nonsuppurative hepatitis. Alpha-toxins and sordellilysin genes from C perfringens and C sordelli isolates, respectively, were detected via polymerase chain reaction. The source of the pathogenic bacteria was sediment within a water basin inside the affected birds' enclosure.

Tracheal Resection in a Secretary Bird ( Sagittarius serpentarius) with Granulomatous, Foreign-body Induced Tracheitis.

A 24-year-old female secretary bird ( Sagittarius serpentarius) was presented with acute, mild dyspnea occurring only during feeding times. Despite initial conservative therapy consisting of antibiotics and antifungal, antiparasitic, and anti-inflammatory drugs, the dyspnea worsened progressively, resulting in severe respiratory distress. Radiographs of the trachea suggested stenosis in the caudal one-third of the trachea. Tracheal endoscopy revealed an obstruction of approximately 90% of the tracheal lumen, in addition to mild suspected aspergillosis of the air sacs. Tracheal resection and anastomosis were performed, during which 1.5 cm of abnormal trachea was removed. Histopathologic examination showed severe granulomatous tracheitis, most likely induced by foreign body material. Respiratory signs resolved immediately postoperatively. Antibiotic and anti-inflammatory therapy continued for another 7 days and the bird was treated with antifungals for a total of 45 days. The bird recovered uneventfully. We encourage tracheal resection and anastomosis for severe tracheal stenosis even in aged, large birds of prey that are managed in large aviaries.

Supression of testicular function in a male Asian elephant (Elephas maximus) treated with gonadotropin-releasing hormone vaccines.

The ability to control testosterone concentrations and sperm production is of great interest in both Asian (Elephas maximus) and African (Loxodonta africana) elephants. GnRH vaccination may pose an alternative to surgical castration. This is a case report of a male Asian elephant treated with two commercial GnRH vaccines (Equity and Improvac). Beginning at the age of 7 yr, the male was vaccinated monthly for 6 consecutive months, then every 6 mo and, finally, every 12 to 24 mo over a period of 6 yr. In order to evaluate the GnRH vaccine as a potential method of immunologic castration, behavioral observations, testosterone level analysis, body weights, ultrasound examinations, and semen collection were part of the routine monitoring of this bull (no. 1) and a half-brother (bull 2) who remained untreated and served as control. The results showed a decrease in serum testosterone concentrations after the second booster. Levels stayed continuously below 5.0 ng/ml within the study period. The combined testicle diameter of 9.03 +/- 0.3 cm prior to treatment had decreased to a size of 6.93 +/- 0.19 cm (P < 0.001) when measured 2 yr later. Accessory sex gland fluid content disappeared and penile atrophy was observed. Semen collections yielded no spermatozoa 1 yr after the initial treatment. Bull 1 showed slowed weight gain as compared to bull 2 and, due to its friendly temperament and the absence of musth, remained in free contact. This report documents the GnRH vaccine as a possible noninvasive and inexpensive method for immune-castration.

Successful nonsurgical artificial insemination and hormonal monitoring in an Asiatic golden cat (Catopuma temmincki).

Since it is reported to be difficult to establish Asiatic golden cat (Catopuma temmincki) breeding pairs in captivity as a result of overaggressive behavior of the male, artificial insemination (AI) may be a desired option by which to achieve pregnancy. This approach was chosen in the present case involving a nulliparous, 6-yr-old female cat that was inseminated transcervically during a naturally occurring estrus, which therefore required only a single general anesthetic procedure. On day 4 of estrus behavior, the male was anesthetized and semen was collected via urethral catheterization (UC) to recover spermatozoa in high concentration followed by electroejaculation (EE) to obtain additional semen and seminal fluid. The fresh UC semen, totaling 180 microl in volume and containing spermatozoa showing 55-70% sperm motility, was inseminated 2.5 hr later via a commercial cat urinary catheter passed through the cervix into the uterus. Immediately afterwards, the EE fraction (100 microl) was inseminated deeply into the dorsal medial fold of the vagina. The GnRH analogue Receptal (0.75 ml, i.m.) was given during anesthesia in an attempt to induce ovulation. Increasing fecal concentrations of progesterone after AI and a significant rise in fecal prostaglandin F2alpha metabolite (PGFM) concentrations (P < 0.0001) from day 45 post-AI indicated that the cat had conceived, and it produced healthy twin cubs after an 84-day gestation.

Gestating for 22 months: luteal development and pregnancy maintenance in elephants.

The corpus luteum, a temporally established endocrine gland, formed on the ovary from remaining cells of the ovulated follicle, plays a key role in maintaining the early mammalian pregnancy by secreting progesterone. Despite being a monovular species, 2-12 corpora lutea (CLs) were found on the elephant ovaries during their long pregnancy lasting on average 640 days. However, the function and the formation of the additional CLs and their meaning remain unexplained. Here, we show from the example of the elephant, the close relationship between the maternally determined luteal phase length, the formation of multiple luteal structures and their progestagen secretion, the timespan of early embryonic development until implantation and maternal recognition. Through three-dimensional and Colour Flow ultrasonography of the ovaries and the uterus, we conclude that pregnant elephants maintain active CL throughout gestation that appear as main source of progestagens. Two LH peaks during the follicular phase ensure the development of a set of 5.4 ± 2.7 CLs. Accessory CLs (acCLs) form prior to ovulation after the first luteinizing hormone (LH) peak, while the ovulatory CL (ovCL) forms after the second LH peak. After five to six weeks (the normal luteal phase lifespan), all existing CLs begin to regress. However, they resume growing as soon as an embryo becomes ultrasonographically apparent on day 49 ± 2. After this time, all pregnancy CLs grow significantly larger than in a non-conceptive luteal phase and are maintained until after parturition. The long luteal phase is congruent with a slow early embryonic development and luteal rescue only starts 'last minute', with presumed implantation of the embryo. Our findings demonstrate a highly successful reproductive solution, different from currently described mammalian models.

The secretory pattern and source of immunoreactive prolactin in pregnant African (Loxodonta africana) and Asian (Elephas maximus) elephants.

The objective of the present study was to define the secretion of prolactin (PRL) in pregnant African and Asian elephants. Levels of immunoreactive (ir-) PRL in serum and placental homogenates were measured by a heterologous radioimmunoassay (RIA) based on an ovine and human RIA system, and the localization of ir-PRL in the placenta was detected by immunohistochemistry using anti-human PRL. Circulating ir-PRL clearly showed a biphasic pattern during pregnancy in African and Asian elephants. Serum levels of ir-PRL started to increase from the 4 - 6th month of gestation and reached the first peak level around the 11-14th month. A second peak of circulating ir-PRL levels was observed around the 18-20th month of gestation followed by an abrupt decline after parturition. In contrast, in a case of abortion of an African elephant, the second peak of ir-PRL was not observed, and the levels remained low for about four months until parturition. The weight of the fetus delivered at the 17th month of gestation was 23.5 kg, which was quite small compared with normal fetuses in previous reports. Ir-PRL was detected in placental homogenates, and immunolocalization was observed in trophoblasts in both the African and Asian elephants, indicating that the placenta is the source of ir-PRL during pregnancy in elephants. The present results clearly demonstrated that circulating ir-PRL shows a biphasic pattern during normal pregnancy and that the placenta appears to be an important source of circulating ir-PRL during pregnancy in both African and Asian elephants.

Role of the double luteinizing hormone peak, luteinizing follicles, and the secretion of inhibin for dominant follicle selection in Asian elephants (Elephas maximus).

Elephants express two luteinizing hormone (LH) peaks timed 3 wk apart during the follicular phase. This is in marked contrast with the classic mammalian estrous cycle model with its single, ovulation-inducing LH peak. It is not clear why ovulation and a rise in progesterone only occur after the second LH peak in elephants. However, by combining ovarian ultrasound and hormone measurements in five Asian elephants (Elephas maximus), we have found a novel strategy for dominant follicle selection and luteal tissue accumulation. Two distinct waves of follicles develop during the follicular phase, each of which is terminated by an LH peak. At the first (anovulatory) LH surge, the largest follicles measure between 10 and 19.0 mm. At 7 ± 2.4 days before the second (ovulatory) LH surge, luteinization of these large follicles occurs. Simultaneously with luteinized follicle (LUF) formation, immunoreactive (ir) inhibin concentrations rise and stay elevated for 41.8 ± 5.8 days after ovulation and the subsequent rise in progesterone. We have found a significant relationship between LUF diameter and serum ir-inhibin level (r(2) = 0.82, P < 0.001). The results indicate that circulating ir-inhibin concentrations are derived from the luteinized granulosa cells of LUFs. Therefore, it appears that the development of LUFs is a precondition for inhibin secretion, which in turn impacts the selection of the ovulatory follicle. Only now, a single dominant follicle may deviate from the second follicular wave and ovulate after the second LH peak. Thus, elephants have evolved a different strategy for corpus luteum formation and selection of the ovulatory follicle as compared with other mammals.