Barbara Drews

Foetal age determination and development in elephants

Elephants have the longest pregnancy of all mammals, with an average gestation of around 660 days, so their embryonic and foetal development have always been of special interest. Hitherto, it has only been possible to estimate foetal ages from theoretical calculations based on foetal mass. The recent development of sophisticated ultrasound procedures for elephants has now made it possible to monitor the growth and development of foetuses of known gestational age conceived in captivity from natural matings or artificial insemination. We have studied the early stages of pregnancy in 10 captive Asian and 9 African elephants by transrectal ultrasound. Measurements of foetal crown–rump lengths have provided the first accurate growth curves, which differ significantly from the previous theoretical estimates based on the cube root of foetal mass. We have used these to age 22 African elephant foetuses collected during culling operations. Pregnancy can be first recognized ultrasonographically by day 50, the presumptive yolk sac by about day 75 and the zonary placenta by about day 85. The trunk is first recognizable by days 85–90 and is distinct by day 104, while the first heartbeats are evident from around day 80. By combining ultrasonography and morphology, we have been able to produce the first reliable criteria for estimating gestational age and ontological development of Asian and African elephant foetuses during the first third of gestation.

The Long Gestation of the Small Naked Mole-Rat (Heterocephalus glaber RÜPPELL, 1842) Studied with Ultrasound Biomicroscopy and 3D-Ultrasonography

The naked mole-rat (Heterocephalus glaber) is one of the two known mammalian species that live in a eusocial population structure. Here we investigate the exceptionally long gestation period of 70 days observed in the mole-rat queen. The course of seven successful pregnancies in two individuals was recorded in a colony of captive naked mole-rats using ultrasound biomicroscopy (UBM) and 3D-ultrasonography. We establish a catalogue of basic reference ultrasound data for this species by describing the ultrasonographic appearance of reproductive organs, calculating growth curves to predict gestational age and defining ultrasonographic milestones to characterize pregnancy stages. Mean litter size was 10.9±2.7, of which 7.2±1.5 survived the weaning period. Mean interbirth interval was 128.8±63.0 days. The reproductive success in our colony did not differ from previously published data. In the queen the active corpora lutea had an anechoic, fluid filled centre. Using UBM, pregnancy could be detected 53 days before parturition. The period of embryonic development is assumed to last until 30 days before parturition. Embryonic resorptions were detected frequently in the queen, indicating that this might be an ordinary event in this species. We discuss the extraordinary long gestation period of this small rodent and postulate that the long gestation is beneficial to both the eusocial structure and longevity. An increased litter size, twice as large as for other rodents of similar size, seemingly compensates for the doubling of pregnancy length. We demonstrate that the lifetime reproductive effort of a naked mole-rat queen is equivalent to the mass of offspring that would be produced if all of the females of a colony would be reproducing.

Early embryo development in the elephant assessed by serial ultrasound examinations

The elephant has an extraordinary long pregnancy, lasting 21 months. However, knowledge on embryo development is limited. To date, only single morphological observations of elephant embryo development associated with placentation are available, all lacking correlation to gestational age. The present study describes morphological characteristics of early embryo development in the elephant with exact biometric staging. Six pregnancies in five Asian and one African elephants with known conception dates were followed by 2D and 3D ultrasound, covering the embryonic period from ovulation to day 116 post-ovulation. The embryonic vesicle was earliest observed was on day 50 p.o. The proper embryo was not detected until day 62 p.o. Embryonic heartbeat was first observed on day 71 p.o. The allantois, which became visible as a single sacculation on day 71 p.o. was subdivided in four compartments on day 76 p.o. By day 95 p.o., head, rump, front and hind legs were clearly distinguished. Between days 95 and 103 p.o. the choriovitelline placenta was replaced by the chorioallantoic placenta. A physiological midgut herniation was transiently present between days 95 and 116 p.o. On the basis of the late appearance of the embryonic vesicle, delayed implantation in the elephant is discussed. The study provides a coherent description of elephant embryonic development, formation of the extraembryonic organs and their role in placenta formation, all of which are of interest for both comparative evolutionary studies and the improvement of assisted reproduction techniques.

Early detection and staging of spontaneous embryo resorption by ultrasound biomicroscopy in murine pregnancy

BackgroundEmbryo resorption is a major problem in human medicine, agricultural animal production and in conservation breeding programs. Underlying mechanisms have been investigated in the well characterised mouse model. However, post mortem studies are limited by the rapid disintegration of embryonic structures. A method to reliably identify embryo resorption in alive animals has not been established yet. In our study we aim to detect embryos undergoing resorption in vivo at the earliest possible stage by ultra-high frequency ultrasound.MethodsIn a longitudinal study, we monitored 30 pregnancies of wild type C57BI/6 mice using ultra-high frequency ultrasound (30-70 MHz), so called ultrasound biomicroscopy (UBM). We compared the sonoembryology of mouse conceptuses under spontaneous resorption and neighbouring healthy conceptuses and correlated the live ultrasound data with the respective histology.ResultsThe process of embryo resorption comprised of four stages: first, the conceptus exhibited growth retardation, second, bradycardia and pericardial edema were observed, third, further development ceased and the embryo died, and finally embryo remnants were resorbed by maternal immune cells. In early gestation (day 7 and 8), growth retardation was characterized by a small embryonic cavity. The embryo and its membranes were ill defined or did not develop at all. The echodensity of the embryonic fluid increased and within one to two days, the embryo and its cavity disappeared and was transformed into echodense tissue surrounded by fluid filled caverns. In corresponding histologic preparations, fibrinoid material interspersed with maternal granulocytes and lacunae filled with maternal blood were observed. In later stages (day 9–11) resorption prone embryos were one day behind in their development compared to their normal siblings. The space between Reichert’s membrane and inner yolk sac membrane was enlarged The growth retarded embryos exhibited bradycardia and ultimately cessation of heart beat. Corresponding histology showed apoptotic cells in the embryo while the placenta was still intact. In the subsequent resorption process first the embryo and then its membranes disappeared.ConclusionsOur results provide a temporal time course of embryo resorption. With this method, animals exhibiting embryo resorption can be targeted, enabling the investigation of underlying mechanisms before the onset of total embryo disintegration.

Ultrasonography of wallaby prenatal development shows that the climb to the pouch begins in utero.

Marsupials have a functional placenta for a shorter period of time compared to that of eutherian species, and their altricial young reach the teats without any help from the mother. We have monitored the short intrauterine development of one marsupial, the tammar wallaby, with high-resolution ultrasound from reactivation of the 100-cell diapausing blastocyst to birth. The expanding blastocyst could be visualized when it had reached a diameter of 1.5 mm. From at least halfway through pregnancy, there are strong undulating movements of the endometrium that massage the expanding vesicle against the highly secretory endometrial surface. These unique movements possibly enhance exchange of uterine secretions and gases between the mother and embryo. There was a constant rate of development measured ultrasonographically from mid-gestation, regardless of when the blastocyst reactivated. Interestingly climbing movements by the fetus began in utero about 3 days before birth, mimicking those required to climb to the pouch.

Ultrasonographically documented early pregnancy loss in an Asian elephant (Elephas maximus).

Early embryonic resorption or fetal loss is known to occur occasionally in captive elephants; however, this has mostly been reported anecdotally. The present study documents the case of a 24-year-old, multiparous Asian elephant cow that suffered embryonic death and resorption at around 18 weeks of gestation. From ovulation onwards, this female was sonographically examined 58 times. Blood was collected twice weekly for progestagen determination via enzyme immunoassay. On Day 42 after ovulation, a small quantity of fluid was detected in the uterine horn, which typically indicates the presence of a developing conceptus. Repeated inspections followed what appeared to be a normal pregnancy until Day 116. However, on Day 124, signs of embryonic life were absent. Progestagen concentrations started declining two weeks later, reaching baseline levels one month after embryonic death. Retrospectively, ultrasound examination revealed several abnormalities in the uterine horn. Besides an existing leiomyoma, multiple small cystic structures had formed in the endometrium at the implantation site and later in the placenta. These pathological findings were considered as possible contributors to the early pregnancy failure. PCR for endotheliotropic elephant herpes virus (EEHV) (which had occurred previously in the herd) as well as serology for other infectious organisms known to cause abortion in domestic animals did not yield any positive results. Although no definitive reason was found for this pregnancy to abort, this ultrasonographically and endocrinologically documented study of an early pregnancy loss provides important insights into the resorption process in Asian elephants.

Ultrasonographic Monitoring of Fetal Development in Unrestrained Bonobos (Pan paniscus) at the Milwaukee County Zoo

The bonobo, Pan paniscus, is one of the most endangered primate species. In the context of the Bonobo Species Survival Plan(®), the Milwaukee County Zoo established a successful breeding group. Although the bonobo serves as a model species for human evolution, no prenatal growth curves are available. To develop growth graphs, the animals at the Milwaukee County Zoo were trained by positive reinforcement to allow for ultrasound exams without restraint. With this method, the well being of mother and fetus were maintained and ultrasound exams could be performed frequently. The ovulation date of the four animals in the study was determined exactly so that gestational age was known for each examination. Measurements of biparietal diameter (BPD), head circumference (HC), abdominal circumference (AC), and femur length (FL) were used to create growth curves. Prenatal growth of P. paniscus was compared with the data of humans and the common chimpanzee, P. troglodytes. With respect to cranial structures, such as BPD and HC, humans have significant acceleration of growth compared with P. paniscus and P. troglodytes. In P. paniscus, growth of AC was similar to HC throughout pregnancy, whereas in humans AC only reaches the level of HC close to term. Growth rate of FL was similar in humans and the two Pan species until near day 180 post-ovulation. After that, the Pan species FL growth slowed compared with human FL. The newly developed fetal growth curves of P. paniscus will assist in monitoring prenatal development and predicting birth dates of this highly endangered species.

Epidemiology, control and management of an EBHS outbreak in captive hares.

Here we describe an outbreak of European brown hare syndrome (EBHS) in a captive hare population. The EBHS outbreak occurred in March 2009, at the beginning of the breeding season. Overall mortality was 53% out of an original population of 61 animals. Animals between five and eleven months showed a significantly higher mortality rate than other age classes. Pregnant females either aborted their foetuses and survived or died pregnant. All foetuses (n=10) of the pregnant hares were PCR positive for EBHSV. Only one offspring born during the outbreak survived. Shortly after the outbreak, the surviving hares developed a specific anti-EBHSV titre between 1:80 and 1:2560, which dropped to 1:10-1:160 nine months later. Hares between one and three years of age developed a significantly higher titre than hares younger than one year or older than four years. Offspring born after the outbreak showed a lower titre of 1:10, indicating passive antibody transfer via placenta and milk. After two months, the titre was not detectable any longer. In December 2009, the captive population was vaccinated against EBHS virus with inactivated virus prepared from the organs of infected hares. The titres after the first vaccination ranged from 1:10 to 1:640, and after the second vaccination from 1:10 to 1:320. To estimate the effect of EBHS on reproduction, we compared the breeding seasons 2008 and 2009. Several possible sources of infection of the colony are discussed, but the definite cause could not be determined.

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.

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.