Linking hunting weaponry to attack strategies in sailfish and striped marlin.

Linking morphological differences in foraging adaptations to prey choice and feeding strategies has provided major evolutionary insights across taxa. Here, we combine behavioural and morphological approaches to explore and compare the role of the rostrum (bill) and micro-teeth in the feeding behaviour of sailfish (Istiophorus platypterus) and striped marlin (Kajikia audax) when attacking schooling sardine prey. Behavioural results from high-speed videos showed that sailfish and striped marlin both regularly made rostrum contact with prey but displayed distinct strategies. Marlin used high-speed dashes, breaking schools apart, often contacting prey incidentally or tapping at isolated prey with their rostra; while sailfish used their rostra more frequently and tended to use a slower, less disruptive approach with more horizontal rostral slashes on cohesive prey schools. Capture success per attack was similar between species, but striped marlin had higher capture rates per minute. The rostra of both species are covered with micro-teeth, and micro-CT imaging showed that species did not differ in average micro-tooth length, but sailfish had a higher density of micro-teeth on the dorsal and ventral sides of their rostra and a higher amount of micro-teeth regrowth, suggesting a greater amount of rostrum use is associated with more investment in micro-teeth. Our analysis shows that the rostra of billfish are used in distinct ways and we discuss our results in the broader context of relationships between morphological and behavioural feeding adaptations across species.

Bronchoalveolar lavage for diagnosis of tuberculosis infection in elephants.

Tuberculosis (TB) has been known to affect elephants for thousands of years. It was put into spotlight when few circus elephants were diagnosed carrying Mycobacterium (M.) tuberculosis. Because of the zoonotic risk and high susceptibility to M. tuberculosis, periodic testing was enacted since, in captive breeding programmes. Presently, trunk wash is the recommended diagnostic procedure for TB. Trunk wash, however, puts the operator at risk, has low sensitivity, and is prone to contamination. Here, bronchoalveolar lavage is described for the first time for TB diagnosis in elephants. Bronchial, trunk and mouth fluids were investigated using bacterial culture, M. tuberculosis complex (MTC)-specific real-time quantitative PCR (qPCR) and mycobacterial genus-specific qPCR for overall presence of mycobacteria or mycobacterial DNA including bacteria or DNA of closely related genera, respectively, in 14 elephants. Neither bacteria of the MTC nor their DNA were identified in any of the elephants. Yet, 25% of the cultures grew non-tuberculous mycobacteria (NTM) or closely related bacterial species. Furthermore, 85% of the samples contained DNA of NTM or closely related bacterial genera. This finding might explain continued false-positive results from various serological tests. From a zoonotic point of view, bronchoalveolar lavage is safer for the testing personal, has higher probability of capturing MTC and, through PCR, identifies DNA NTM in elephants. Yet, necessary endoscopic equipment, animal sedation and access to a TB reference laboratory might pose challenging requirements in remote conditions in some elephant range countries.

Cryopreservation of captive roe deer (Capreolus capreolus) semen.

To address the need to preserve current genetic diversity before it is lost forever; further studies to adapt assisted reproductive technologies to various endangered species are needed, among other things. Roe deer (Capreolus capreolus), an over abundant wild deer, can serve as model species to develop or improve sperm cryopreservation of threatened or endangered deer species. The aim of this study was to compare the ability of three diluents (Berliner Cryomedium [BC]; Tris, citric acid, glucose [TCG]; TES, Tris, glucose) to support chilling, cryopreservation (with 5% glycerol; G) and postthaw incubation (at 22 °C and 37 °C) of roe deer spermatozoa collected by electroejaculation. Berliner Cryomedium was the diluent that better preserved roe deer spermatozoa during refrigeration, able to maintain motility for at least 14 days, longer than the other extenders. BC + G was the extender of choice for cryopreservation, showing higher viability compared with TCG + G (66.7 ± 3.4 vs. 54.5 ± 6.5; P < 0.05) and higher level of acrosome integrity compared with TES, Tris, glucose + G (79.4 ± 3.4 vs. 67.9 ± 5.0; P < 0.05). Maintaining the samples at 22 °C after thawing presented higher values in various parameters compared with 37 °C. The knowledge gained through this study can potentially act as a preliminary step toward development of new protocols to help increase the reproductive success of biologically similar, yet endangered, wild species.

Study of Age-Dependent Structural and Functional Changes of Mitochondria in Skeletal Muscles and Heart of Naked Mole Rats (Heterocephalus glaber).

Morphometric analysis of mitochondria in skeletal muscles and heart of 6- and 60-month-old naked mole rats (Heterocephalus glaber) revealed a significant age-dependent increase in the total area of mitochondrial cross-sections in studied muscle fibers. For 6- and 60-month-old animals, these values were 4.8 ± 0.4 and 12.7 ± 1.8%, respectively. This effect is mainly based on an increase in the number of mitochondria. In 6-month-old naked mole rats, there were 0.23 ± 0.02 mitochondrial cross-sections per µm2 of muscle fiber, while in 60-month-old animals this value was 0.47 ± 0.03. The average area of a single mitochondrial cross-section also increased with age in skeletal muscles - from 0.21 ± 0.01 to 0.29 ± 0.03 µm2. Thus, naked mole rats show a drastic enlargement of the mitochondrial apparatus in skeletal muscles with age due to an increase in the number of mitochondria and their size. They possess a neotenic type of chondriome accompanied by specific features of mitochondrial functioning in the state of oxidative phosphorylation and a significant decrease in the level of matrix adenine nucleotides.

Embryonic resorption in context to intragestational corpus luteum regression: a longitudinal ultrasonographic study in the European brown hare (Lepus europaeus PALLAS, 1778).

Embryonic resorption is frequently observed in polytocous mammals. Often it occurs as partial litter resorption affecting only single conceptuses of a whole litter. The aim of the study was to describe the incidence and morphology of embryonic resorption in the European brown hare (Lepus europaeus). In 154 pregnancies viable conceptuses, conceptuses undergoing resorption and CL of pregnancy were ultrasonographically monitored during the entire gestation period. Resorptions were classified into (1) "pre-implantation resorptions," (2) "peri-implantation resorptions," and (3) "post-implantation resorptions." The incidence of resorption in the pre-implantation period was 9%, in the peri-implantation period 9%, and in the post-implantation period 24%. Post-implantation resorptions were found up to late pregnancy stages when fetal development was already in progress. The highest daily incidence of resorption was on Day 8 of the 42-day pregnancy. In 91% of the cases, the regression of one CL was observed, while an embryo was undergoing resorption at the same time. The number of resorptions did not significantly differ from the number of CL in regression during gestation, suggesting an interesting one-resorption-to-one-regression relationship. The ultrasonographic appearance of the luteal regression during pregnancy was similar to the morphology characteristic for postpartal luteolysis.

Cryopreservation of aoudad (Ammotragus lervia sahariensis) sperm obtained by transrectal ultrasound-guided massage of the accessory sex glands and electroejaculation.

This study examines (1) the effectiveness of transrectal, ultrasound-guided massage of the accessory sex glands (TUMASG) combined with electroejaculation for obtaining aoudad (Ammotragus lervia sahariensis) sperm samples for cryopreservation, and (2) the effectiveness of a Tris-citric acid-glucose-based medium (TCG; usually used for freezing ibex sperm) and a TES-Tris-glucose-based medium (TTG; typically used in the cryopreservation of mouflon sperm) as sperm extenders. After TUMASG, just one to three electrical pulses were required for ejaculation to occur in five of the six animals studied; one ejaculated after TUMASG alone. Transrectal, ultrasound-guided massage of the accessory sex glands would therefore appear to be useful in obtaining sperm samples from this species, requiring few subsequent electrical electroejaculation stimuli and sometimes none at all. After thawing, the membrane integrity (assessed by nigrosin-eosin staining) of sperm extended with TTG was greater than that of sperm extended with TCG (P < 0.05). The total percentage of sperm showing an intact acrosome, as assessed by fluorescein isothiocyanate-conjugated peanut (Arachis hypogea) agglutinin, was also higher in the TTG-extended sperm (P < 0.05), and the percentage of dead sperm with a damaged acrosome was lower (P < 0.05). No differences were seen between TCG and TTG in terms of apoptotic manifestations (DNA damage, caspase activity, mitochondrial membrane potential, and plasmalemma stability). Therefore, TTG appears to be a better extender than TCG for cryopreserving aoudad sperm.

Canine tarsal architecture as revealed by high-resolution computed tomography.

Central tarsal bone (CTB) fractures are well documented and are a subject of increasing importance in human, equine and canine athletes although the mechanism of these fractures in dogs is not fully understood and an extrapolation from human medicine may not be accurate. This study reports the use of high-resolution computed tomography (CT) of 91 tarsal joints from 47 dogs to generate a more detailed in situ anatomical description of the CTB architecture in order to obtain a better understanding of the pathogenesis of CTB fractures in this species. The dogs studied represented a wide range of ages, breeds and levels of habitual physical activity and the angles of the tarsal joints studied ranged between maximal flexion (16.4°) and maximal extension (159.1°). Regardless of tarsal angle, the CTB articulated with the calcaneus exclusively at the level of its plantar process (PPCTB) in all dogs. The PPCTB presented two distinct parts in all dogs, a head and a neck. The calcaneus tended to rely on the PPCTB neck during flexion and on the PPCTB head during extension. This study describes new tarsal elements for the first time, including the calcaneal articular process, the fourth tarsal bone plantar articular process and the talar plantar prominence of the CTB. Based on calcaneo-PPCTB architecture, it is postulated that the PPCTB is a keystone structure and that at least some of CTB fractures in dogs could either commence at or are induced at this level due to the impingement forces exercised by the calcaneus.

Enriching the captive elephant population genetic pool through artificial insemination with frozen-thawed semen collected in the wild.

The first successful AI in an elephant was reported in 1998, using fresh semen. Since then almost 40 calves have been produced through AI in both Asian and African elephants worldwide. Following these successes, with the objective of enriching the captive population with genetic material from the wild, we evaluated the possibility of using frozen-thawed semen collected from wild bulls for AI in captivity. Semen, collected from a 36-yr-old wild African savanna elephant (Loxodonta africana) in South Africa was frozen using the directional freezing technique. This frozen-thawed semen was used for four inseminations over two consecutive days, two before and two after ovulation, in a 26-yr-old female African savanna elephant in Austria. Insemination dose of 1200 × 10(6) cells per AI with 61% motility resulted in pregnancy, which was confirmed through ultrasound examination 75, 110 and 141 days after the AI procedure. This represents the first successful AI using wild bull frozen-thawed semen in elephants. The incorporation of AI with frozen-thawed semen into the assisted reproduction toolbox opens the way to preserve and transport semen between distant individuals in captivity or, as was done in this study, between wild and captive populations, without the need to transport stressed or potentially disease-carrying animals or to remove animals from the wild. In addition, cryopreserved spermatozoa, in combination with AI, are useful methods to extend the reproductive lifespan of individuals beyond their biological lifespan and an important tool for genetic diversity management and phenotype selection in these endangered mammals.

Estrus induction in white rhinoceros (Ceratotherium simum).

The estrous cycle length in the white rhinoceros (Ceratotherium simum) is either 4 or 10 wk. The cause(s) for this variation as well as the poor fertility rate in captivity remains under debate in this species. Most captive adult white rhinoceros undergo long anovulatory periods without luteal activity which are considered a major reason for their low reproductive rate. In this study, the synthetic progestin chlormadinone acetate (CMA) was tested in combination with hCG or the GnRH analogue deslorelin for its efficiency to induce ovulation in fourteen females without luteal activity and in three, regular cycling females. HCG (N = 12), injectable GnRH analogue (N = 8) and GnRH analogue implants (N = 15) were given to induce ovulation after CMA treatment. Treatment success was determined using both transrectal ultrasonography and progesterone metabolite EIA analysis. A preovulatory sized follicle (3.5 ± 0.1 cm) or a corpus luteum (5.1 ± 0.7) was present on the ovary one day after induction in 93.1% of the treatments. Despite this high rate of ovarian response, ovulation rate differed between the study groups. The ovulation rate for hCG, injectable GnRH analogue and GnRH analogue implants was 66.7%, 62.5% and 93.3%, respectively. Ovulation rate in cyclic females treated with GnRH implants was 100% (6/6) compared with 89% (8/9) in females without luteal activity receiving the same treatment. The length of the estrous cycle when induced with hCG was 4 wk (85.7%). The estrous cycle when induced with GnRH analogue was predominantly 10 wk long. Two females without luteal activity treated with GnRH became pregnant. In conclusion, CMA in combination with GnRH analogue implants was highly effective to induce ovulation in white rhinoceroses and thus can contribute to efforts aimed at increasing natural mating and reproductive rates in the captive white rhinoceros population.

Embryo retrieval after hormonal treatment to control ovarian function and non-surgical artificial insemination in African lions (Panthera leo).

Assisted reproduction technologies are essential for propagating endangered wild felids. Artificial insemination (AI) has been reported in several wild feline species, but pregnancy rates are low, partially owing to failures of current hormonal stimulation protocols. Therefore, this study describes the application of reliable methods to monitor ovarian activity and the development of an effective hormonal protocol to induce oestrus and ovulation in African lions. Application of porcine FSH and porcine LH was shown to be effective for inducing follicular growth and ovulation, and this regimen appeared to be superior to protocols described earlier in terms of ovulation and fertilization rates. Furthermore, non-surgical AI was performed successfully in lions, and uterine-stage embryos were collected and cryopreserved. African lions may serve as a valuable model to develop assisted reproduction for propagation of relic zoo populations in the critically endangered Asian lion or Barbary lion.

In vivo tissue sampling of embryonic resorption sites using ultrasound guided biopsy.

In the polytocous European brown hare (Lepus europaeus) more than 23% of all successful implantations undergo embryonic resorption. The objective of the study was to establish a minimally invasive ultrasound guided biopsy technique to collect embryonic resorption tissue in vivo. The sampled material was genetically analysed to determine paternity and the sex of the embryo. Female hares were either mated or artificially inseminated and pregnancy was confirmed by ultrasound on day six post ovulation. Subsequent embryonic development was ultrasonographically monitored on a regular basis to detect embryos undergoing resorption. Cell material of the resorption site was collected under ultrasonographic control via transabdominal biopsy of the placenta or aspiration of resorption fluid. To avoid breathing movements during the biopsy, the animals were intubated and a short apnoea was evoked by assisted ventilation. The presence of embryonic cells in the biopsy material was confirmed by microsatellite analysis in 11 of the fluid samples (n = 28) and six of the placental samples (n = 8). The lower success rate in the fluid samples was attributed to the abundance of maternal cells which was confirmed by the analysis of fluid sample smears. Male sex of the embryos undergoing resorption was detected by SRY analysis for ten of the fluid samples and for one of the placental samples. The two biopsy techniques did not have any negative impact on the prenatal development of the healthy siblings nor did it influence the future breeding performance of the females that were biopsied.

Dystocia and fetotomy associated with cerebral aplasia in a greater one-horned rhinoceros (Rhinoceros unicornis).

The captive greater one-horned rhinoceros population consists of 176 animals. Since 1971, a total of 226 calves were born into this captive population. However, 24% of the offspring born were either stillborn or did not survive the first 3 months. The causes for this high rate of stillbirth and neonate mortality have not yet been documented. Here, we report on the veterinary management of a dystocia and foetotomy resulting from a malpositioned greater one-horned rhinoceros foetus. The dead foetus presented with a forelimb flexed at the shoulder joint, with all other joints extended. The foetus was dissected into five parts and extracted during two anaesthesias on two consecutive days. The dam recovered fully and came into oestrous 31 days after surgery. Post-mortem and CT examination of the malformed foetal head revealed cranioschisis with cerebral aplasia and cerebellar hypoplasia. The cerebral aplasia presented here and in other recent cases suggests that neural tube defects and cranial malformations may be associated with more captive rhinoceros stillbirths than previously considered. Epidemiologic studies of these phenomena and possible nutritional deficiencies or hereditary defects are warranted.

Collection and preservation of pygmy hippopotamus (Choeropsis liberiensis) semen.

Knowledge about the reproduction of the endangered pygmy hippopotamus is almost non-existent. This study takes the first step toward changing this by devising a protocol for the collection, evaluation, and short-term preservation of semen of this endangered species. Semen was collected successfully from seven bulls by electroejaculation, using a specially designed rectal probe. Mean +/- SEM values of native sperm parameters from combined best fractions were: motility-80.0 +/- 4.1%, concentration-2421 +/- 1530 x 10(6) cells/mL, total collected cell number-759 +/- 261 x 10(6) cells, intact acrosome-87.8 +/- 1.2%, intact morphology-52.7 +/- 4.3%, and, for some, hypoosmotic swelling test-79.3 +/- 4.4% and seminal plasma osmolarity-297.5 +/- 3.3 mOsm. Seven different extenders were tested for sperm storage under chilling conditions: Berliner Cryomedium (BC), Biladyl, modification of Kenney modified Tyrode's medium (KMT), MES medium, Androhep((R)), boar M III() extender and Human Sperm Refrigeration Medium. While differences between males were apparent, the BC was consistently superior to all other extenders in sperm motility and facilitated storage for 7 d with up to 30% motility and some motility even after 3 weeks. With this knowledge in hand, the obvious two directions for future research are to conduct artificial insemination and to develop a technique for sperm cryopreservation.

Seminoma in a southern black rhinoceros (Diceros bicornis minor): diagnosis, surgical management and effect on fertility.

A testicular mass was identified by ultrasonography performed during a routine reproductive evaluation of an adult male southern black rhinoceros (Diceros bicornis minor). Histological examination of a testicular biopsy supported a presumptive diagnosis of testicular neoplasia. Hemi-castration was performed to excise the affected testis and a pathological diagnosis of a seminoma was made. Assessment of semen suggested reduced fertility as a consequence of the neoplastic process, but hemi-castration prevented further growth and metastasis of the tumour and ensured the animal's breeding potential. This is the second documented case of a seminoma in a rhinoceros species and the first case in a black rhinoceros.

Ovarian superstimulation, transrectal ultrasound-guided oocyte recovery, and IVF in rhinoceros.

Numerous reports on reproductive pathology in all rhinoceros species illustrate the abundance of female infertility in captive populations. In infertile rhinoceroses, oocyte collection and embryo production could represent the best remaining option for these animals to reproduce and to contribute to the genetic pool. We report here on superstimulation, repeated oocyte recovery, and attempted in vitro fertilization (IVF) in white and black rhinoceroses. Four anestrous rhinoceroses (two white, two black) with unknown follicular status were treated with gonadotropin-releasing hormone analogue, deslorelin acetate, for 6 to 7 d. Number and size of follicles in superstimulated females was significantly higher and larger compared with those in nonstimulated anestrous females (n=9). Ovum pick-up was achieved by transrectal ultrasound-guided follicle aspiration. Up to 15 follicles were aspirated per ovary. During six ovum pick-ups, a total of 29 cumulus-oocyte complexes (COCs) were harvested with a range of 2 to 9 COCs per collection. No postsurgical complications were noted on the rhinoceros ovaries using this minimally invasive approach. Various in vitro maturation (IVM) and IVF protocols were tested on the collected COCs. Despite the low total number of COCs available for IVM and IVF in this study, we can report the first rhinoceros embryo ever produced in vitro. The production of a 4-cell embryo demonstrated the potential of transrectal ultrasound-guided oocyte recovery as a valuable tool for in vitro production of rhinoceros embryos from otherwise infertile females.

Non cat-like ovarian cycle in the Eurasian and the Iberian lynx - ultrasonographical and endocrinological analysis.

The Iberian lynx is considered the most endangered felid species. Therefore, an ex situ conservation program was initiated to protect this species from extinction. Additional knowledge on lynx reproduction biology and reliable methods for reproductive monitoring are important for developing a captive breeding program. The aim of this study in lynx was to implement transrectal ultrasonography to visualize ovarian structures (follicles, corpora lutea) and to assess ovarian activity in addition to analysis of serum progesterone and oestradiol. Because of limited access to Iberian lynxes, the less-endangered Eurasian lynx and bobcat were also studied in this comparative study. Recent endocrinological studies based on faecal and urinary progesterone and oestrogen metabolites revealed that steroid profiles in both these species were alike and did not follow the typical pattern of other felids. Pregnancy diagnosis was not possible, since progesterone concentrations did not differ between pregnant and pseudopregnant animals. Progesterone was also detected after parturition as well as after weaning until the onset of a new oestrous cycle. In the present study, the presence of corpora lutea during the non-breeding season was confirmed by ultrasonography and by elevated serum levels of progesterone averaging 3.56 +/- 1.3 ng/ml in Eurasian and 6.1 +/- 0.26 ng/ml in Iberian lynx, respectively. The ultrasonographical findings on the ovarian structures suggest strongly that corpora lutea developed after ovulation stay active until November and regress before the onset of the next oestrus.

Gonadal activity in male and female captive fossas (Cryptoprocta ferox) during the mating season.

The fossa is an endangered, mongoose-like carnivore species endemic to Madagascar with a breeding season (BS) in the southern hemisphere spring. For the present study, faecal samples of captive fossas were collected for over 1 year for five males and four non-pregnant females, and for two pregnant animals during the reproductive period. The goal was to assess gonadal activity using non-invasive hormone measurements of faecal testosterone (T) and gestagen metabolites using assays previously established in our laboratory and further validated in this study. All study animals were housed in northern hemisphere zoos. In males, the seasonal T metabolite profile revealed a peak in spring (March). High performance liquid chromatography (HPLC) analysis was used to characterize the faecal hormone metabolite composition. The highest immunoreactivity was detected in the position of dihydrotestosterone, whereas native T was not detected. In the two pregnant females, gestagen metabolite concentrations increased 4-9 days after the initial matings and remained elevated throughout gestation with concentrations dramatically higher than those of the non-pregnant females during the BS. In these females, gestagen metabolite analyses did not reveal a seasonal pattern similar to the males. The HPLC-analysis revealed that the major proportion of immunoreactivity was associated with an unknown metabolite, whereas native progesterone was undetectable. The seasonal hormone pattern of male fossas gives proof of the reproductive seasonality of this species. The elevated 5alpha-pregnan-3beta-ol-20-one levels in pregnant animals allows for the characterization of pregnancy in the fossa based on analysis of faecal steroid metabolite concentrations.

Pregnancy monitoring in dogs and cats using 3D and 4D ultrasonography.

Three-dimensional (3D)/four-dimensional (4D) volume ultrasound is an established method in human medicine that offers various options for analysing and presenting ultrasound volume data. However, the successful application of the different 3D/4D imaging modalities in pregnant dogs and cats has not yet been reported in the literature. The main reasons for this are: (1) the high costs of 3D/4D ultrasound systems, (2) operation difficulties due to high breathing frequency in non-sedated animals and (3) the missing specific knowledge in veterinary medicine concerning how to perform high-quality volume scans. Automatically acquired ultrasound volume data sets were generated with two different ultrasound systems: the portable Voluson i and the stationary Voluson Expert 730. Different 3D/4D imaging modalities were tested in regard of their practicability in pregnancy monitoring in dogs and cats. Nine different volume imaging modalities were applied using the saved files. For the presentation of the static 3D volume data sets, we used the multiplanar, niche, surface, transparency, glass body, inversion, volume calculation and tomographic ultrasound imaging modes. For the dynamic 4D data, the surface and glass body modes were applied. By changing the human standard settings to the requirements of small animal anatomy, it was found that 3D/4D ultrasound has great potential for the characterization of pregnancy in queens and bitches. The 3D/4D technology offered advanced information about pregnancy status and birth prediction and improved the diagnostic confidence. By using standardized examination protocols, 3D/4D ultrasound will allow a reduction in examination time by generating even more relevant information. These benefits, combined with possible future cost reduction of commercial ultrasound systems, might lead to frequent utilization in routine pregnancy diagnostic and birth management in small animal practice.

Artificial insemination of captive European brown hares (Lepus europaeus PALLAS, 1778) with fresh and cryopreserved semen derived from free-ranging males.

This study aimed to establish artificial insemination (AI) protocols to predictably initiate pregnancy during the breeding season in the European brown hare (EBH) (Lepus europaeus PALLAS, 1778). Semen was collected from seven captive and eight free-ranging males by means of electroejaculation. Semen from the free-ranging males was cryopreserved using directional freezing. Total motility/integrity of fresh and frozen-thawed semen was 91.6%/87.7% and 46.9%/53.8%, respectively. Ovulation was induced in ultrasonographically preselected females using a gonadotropin-releasing hormone analogue. Each female was inseminated with 1 mL fresh (Group A, n=16) or frozen-thawed semen (Group B, n=9) at a concentration of 100x10(6) spermatozoa/mL. The use of ultrasonography (10 to 22 MHz) confirmed the intracervical semen deposit, the success of artificial ovulation induction (formation of postovulatory corpus luteum), and permitted the monitoring of individual pregnancies. Although sperm motility/integrity was significantly different between groups, no significant difference was detected in conception rates (A, 87.50%; B, 77.78%). Because of embryonic resorption, there was a slight difference in fertility rate between groups (A, 62.5%; B, 77.78%). Overall, AI in captive EBH using fresh and frozen-thawed semen achieved successful fertility rates. Long-term cryopreserved semen was used to bring new genetic material from the wild into a genetically limited captive population without extensive animal transport. Therefore, AI has the potential to enhance breeding programs for EBH especially when cryopreserved semen from wild donors is used.