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1Department of Microbiology, Immunology, and Pathology, Colorado State University
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Transabdominal ultrasound is described as an effective, noninvasive means for assessing reproductive status in Reeves' muntjac deer. These methods can be used to achieve early pregnancy diagnosis and to evaluate fetal viability. Future applications of this technique include estimation of gestational age and effects of maternal disease on fetal development.
Walton, K. D., McNulty, E., Nalls, A. V., Mathiason, C. K. Transabdominal Ultrasound for Pregnancy Diagnosis in Reeves' Muntjac Deer. J. Vis. Exp. (83), e50855, doi:10.3791/50855 (2014).
Reeves' muntjac deer (Muntiacus reevesi) are a small cervid species native to southeast Asia, and are currently being investigated as a potential model of prion disease transmission and pathogenesis. Vertical transmission is an area of interest among researchers studying infectious diseases, including prion disease, and these investigations require efficient methods for evaluating the effects of maternal infection on reproductive performance. Ultrasonographic examination is a well-established tool for diagnosing pregnancy and assessing fetal health in many animal species1-7, including several species of farmed cervids8-19, however this technique has not been described in Reeves' muntjac deer. Here we describe the application of transabdominal ultrasound to detect pregnancy in muntjac does and to evaluate fetal growth and development throughout the gestational period. Using this procedure, pregnant animals were identified as early as 35 days following doe-buck pairing and this was an effective means to safely monitor the pregnancy at regular intervals. Future goals of this work will include establishing normal fetal measurement references for estimation of gestational age, determining sensitivity and specificity of the technique for diagnosing pregnancy at various stages of gestation, and identifying variations in fetal growth and development under different experimental conditions.
Ultrasonographic examination is a well-described method of pregnancy diagnosis in cattle1, sheep2,3, goats4, horses5, companion animals6,7, and other domestic animal species. This imaging modality has also been identified as a valuable tool for pregnancy detection and fetal age estimation in several species of captive cervids, including fallow deer8-11, red deer12-16, Hokkaido sika deer17, and reindeer18,19. In many cases, ultrasonography has produced higher accuracy rates than other mechanisms of pregnancy detection, such as serum progesterone or pregnancy-associated glycoprotein analysis9,18. Furthermore, ultrasonographic visualization of the reproductive tract can provide additional information regarding fetal viability, gestational age, and identification of developmental abnormalities that would otherwise be difficult to assess. The utility of ultrasonography for evaluating reproductive status suggests that this methodology should be adapted to other species in which active breeding programs are used.
Reeves' muntjac deer (Muntiacus reevesi) are a small cervid species native to southeast Asia20. While the reproductive cycle of these animals is not well-characterized in their native territories, studies of feral populations in parts of southern England have improved understanding of their breeding patterns20. Unlike most cervids, Reeves' muntjac appear to breed year-round, with no apparent seasonal fawning peaks20-22. Female muntjac typically give birth to a single fawn following a 210-day gestation period, and reliably enter a postpartum estrus within 24 hr20-22. Does that do not conceive during this postpartum period will return to estrus approximately 24-25 days later20. In captive situations, reproduction is typically achieved by housing does with one or more intact male. This system produces satisfactory conception rates and requires little technical skill for personnel, however exact breeding dates are difficult, if not impossible, to determine.
Muntjac are commonly used in cytogenetic studies due to the wide range of diploid chromosome numbers and high rate of karyotypic diversification among the different species23. These animals are also currently being investigated as a model of prion disease pathogenesis and transmission, with particular interest placed on the study of vertical transmission of chronic wasting disease (CWD) in cervids. The importance of the muntjac in a number of different research applications suggests that improved reproductive technologies should be established to complement current investigative procedures. Specifically, noninvasive mechanisms to assess the gestational period could potentially elucidate critical variations in reproductive performance and fetal growth and development under different experimental conditions. Furthermore, the development of real-time fetal measurement references would be potentially valuable in estimating gestational age in situations where breeding dates cannot be determined. Previous studies have described mechanisms for using fetal measurements, such as crown-rump length (CRL), chest depth (CD), and head length (HL) to estimate the approximate stage of gestation in cervid species11-14, 16,17. Additionally, some of these studies have also established guidelines describing when specific developmental milestones, such as skeletal mineralization or fetal heartbeat, can be identified8-19. While these data may be useful to help guide reproductive ultrasound analysis in the muntjac, most of the other cervid species studied have much larger body size and longer gestational periods, reducing the translational value of these studies for muntjac applications.
The protocol outlined here describes transabdominal ultrasonography of the female muntjac for the purposes of diagnosing and monitoring pregnancy. Successful execution of this protocol can facilitate early pregnancy detection and evaluation of fetal growth and development. This technique has valuable applications in the conduct of studies investigating reproduction, in utero development, or vertical transmission of infectious diseases in a small cervid model and may also be useful for clinical purposes in captive breeding operations.
1. Procedural Preparation
All animal procedures described have been reviewed and approved by the Institutional Animal Care and Use Committee at Colorado State University, an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC), Intl. accredited institution.
2. Transabdominal Reproductive Ultrasound Examination
3. Obtaining Fetal Measurements
Note: Gestational age in many domestic animal species can be estimated using established reference ranges for fetal measurements obtained from ultrasound examination. The most commonly used measurements include straight crown-rump length (SCRL), chest depth (CD), and head length (HL)17. While sufficient data has not yet been collected to develop reliable gestational age guidelines in Reeves' muntjac deer, measurements may be collected in order to begin establishing these references. Fetal heart rate (FHR) has been shown in other species of cervid and ruminant to increase linearly as gestation progresses to a certain point (typically around day 60 of gestation), at which time the heart rate begins to decrease17. FHR may be used to estimate gestational age in conjunction with other ultrasound observations, however it is more commonly used as a measure of fetal viability17,24. FHR has been calculated in Reeves' muntjac as early as 35 days post-observed mating.
4. Procedural Conclusions and Recovery
Using the protocol described here, pregnancy can be diagnosed and monitored in Reeve's muntjac deer with minimally invasive techniques. The earliest time point that pregnancy could be detected by the authors was approximately 35 days following observed mating behavior.
In the early stages of gestation, pregnancy was diagnosed by visualizing a fluid-filled uterus identified as an anechoic space in the caudal abdomen. Within the uterus, the developing embryo appeared as a hyperechoic, ovoid structure with few identifiable features (Figure 1). As pregnancy progressed, fetal structures became apparent and a conclusive diagnosis could be made following the observation of skeletal mineralization and fetal heartbeat (Figures 2 and 3).
Fetal measurements can be obtained and used to establish reference guidelines for estimating gestational age in cases where an exact breeding date is not known, although we have not yet fully developed this system in the muntjac. Straight crown-rump length (SCRL), HL, and CD are standard fetal measurements that are commonly used to estimate fetal age in many species17, and these measurements were obtained from ultrasound images of muntjac pregnancies using the protocol described here. All three of these measurements can be obtained from the same properly positioned sagittal image (Figures 2 and 3), but separate pictures can be used if the entire fetus is not able to be captured in a single sagittal view. In the future, these techniques can also be used to identify variations in growth and development observed under different experimental conditions and to determine fetal viability.
Figure 1. Ultrasound image demonstrating early pregnancy in a Reeves' muntjac deer. The doe in this study had been housed with an intact male muntjac for 46 days, however an exact breeding date is not known. Fetal features are not distinguishable at this stage of gestation.
Figure 2. Ultrasound image of the same pregnancy depicted in Figure 1, 91 days following introduction of the doe to the buck. (CD - chest depth, HL - head length, P - placentome, SCRL - straight crown-rump length).
Figure 3. Ultrasound image of a second pregnancy demonstrating appropriate positioning for obtaining fetal measurements. Image taken 91 days following introduction of the doe to the buck. (CD - chest depth, HL - head length, P - placentome, SCRL - straight crown-rump length).
This protocol describes the application of transabdominal ultrasonography for pregnancy diagnosis in Reeves' muntjac deer. Consistent with other ultrasound studies, appropriate equipment and technical proficiency are imperative to a successful reproductive examination of the muntjac doe. Careful review of basic mammalian anatomy, the physical principles of diagnostic ultrasound, and the typical ultrasonographic appearance of the abdominal viscera will aid in the reduction of mistaken diagnoses. Furthermore, selection of appropriate transducers and familiarity with the instrumentation and scanner controls is critical for obtaining high-quality images and accurate interpretation.
For many inexperienced ultrasonographers, imaging artifacts are a common cause of poor image resolution and inaccurate assessment of structures. Reverberation at the skin-transducer interface is one of the most frequently encountered artifacts in veterinary abdominal ultrasound, and can be significantly reduced by eliminating the presence of gas between the two surfaces. Adequate hair removal and the generous application of acoustic gel to the skin should be sufficient to minimize the impact of this artifact. Discussions of other imaging artifacts can be found in various texts of human and veterinary ultrasonography, and will therefore not be discussed in further detail here; however, a general knowledge of these phenomena will allow the ultrasonographer to control them more effectively24.
If conducting a reproductive examination, it should be understood that the uterus may be difficult to visualize in a normal, nonpregnant animal. In early pregnancy, the lumen of the uterus may become identifiable as a hypoechoic to anechoic space as the gestational sac forms. At this stage in the muntjac, the uterus can typically be found by scanning at the level of, or slightly caudal to, the mammary glands. Prior to the skeletal mineralization or the appearance of a fetal heartbeat, pregnancy may be misdiagnosed in the presence of other reproductive conditions causing fluid to accumulate in the uterus, which may include pathology such as pyometra, hydrometra, or mucometra. Ultrasound diagnoses should be made in conjunction with a complete clinical assessment of the animal prior to sedation for the procedure. In early gestation, the conceptus will appear as a spherical to ovoid, hyperechoic structure within the amniotic sac. Specific developmental time points are not well-characterized in this species; however skeletal mineralization and fetal cardiac motion appear to be consistently detectable at approximately 8 weeks post-mating.
Under ideal conditions, transabdominal ultrasound can be an incredibly useful tool for evaluation of reproductive structures in many species, including Reeves' muntjac. The value of this technique, however, is highly dependent upon the specific capabilities of the animals' housing facilities. Unlike more passive mechanisms of pregnancy detection, such as visual observation of mating behavior and subsequent abdominal distention of the doe, ultrasound requires physical and/or chemical restraint of the animal for a period of time lasting 5-10 min or longer. In many facilities, this is not possible or practical, and the necessity of the examination should be considered based on the specific objectives of the study. In addition, certain physiological or pathological states may suggest that ultrasonography is contraindicated in certain animals due to the potentially detrimental effects of distress related to capture and sedation. It is important to assess the suitability of each animal for ultrasound examination prior to initiating the study, and a veterinarian should be consulted in the planning stages of these projects.
Current practices for pregnancy detection in Reeves' muntjac and other wildlife species are typically passive in nature (observation of mating behavior and abdominal distention in does). Serum progesterone or pregnancy-associated glycoprotein levels can be measured to confirm the diagnosis18,25, however the results of these assays do not provide any information regarding stage of gestation or fetal viability. Ultrasound offers the benefit of permitting real-time assessment of reproductive status with accuracy similar to or greater than that of serum hormone assays18, while maintaining minimal invasiveness and distress to the animals. In operations where the breeding date is difficult or impossible to determine, ultrasound may provide a more accurate mechanism by which to estimate stage of gestation than hormonal assays, even in the absence of published gestational tables. Furthermore, physical and chemical restraint for ultrasound examination appears to cause minimal distress to healthy muntjac and provides an ideal opportunity to perform a thorough physical examination and collect additional samples (blood, urine, etc.) for general health assessments.
Gestational age estimation through ultrasonographic measurements has been established in many species, including cattle1, sheep2,3, goats4, and horses5, and has been described in a limited number of cervid species as well8-19. In the absence of exact breeding dates or established gestation tables, fetal age of Reeves' muntjac can only be roughly estimated based on fetal development parameters outlined for other species. A specific goal of the work described here is to establish more formal guidelines for fetal aging in this species. With sufficient normative data in place, future applications of ultrasonography in this colony will include identifying abnormalities in fetal development in the presence of different experimental variables and assessing fetal viability. In addition, sensitivity and specificity of transabdominal ultrasound can be determined and compared to other commonly employed mechanisms for detecting pregnancy in cervid species.
The authors have nothing to disclose.
The authors wish to thank Jeanette Hayes-Klug and Kelly Anderson for management of the muntjac colony, Dr. Alan Young for introducing us to the muntjac and Mr. Cleve Tedford of Cervid Solutions Inc. for providing the seed muntjac for this project. This work was supported by NIH grant R01AI093634.
|Butorphanol tartrate (Torbugesic) (0.45 mg/kg)||Fort Dodge Animal Health, Fort Dodge, Iowa||NADA 135-780|
|Azaperone tartrate (0.035 mg/kg)||ZooPharm|
|Medetomidine HCl (0.04 mg/kg)||ZooPharm|
|Ultrasound contact gel||Medline, Mundelein, Illinois||MDS092005|
|Atipamezole hydrochloride (Antisedan) (0.25 mg/kg)||Orion Corporation, Espoo, Finland||NADA 141-033|
|V-trough (optional)||N/A||N/A||Animal may be supported in dorsal recumbency on a standard examination table or other surface without the use of a V-trough|
|Electrical clippers or razor blades for hair removal||Oster, McMinnville, Tennessee||78005-140|
|Ultrasound system: Ibex Pro portable ultrasound||E.I. Medical Imaging, Loveland, CO||Ibex Pro|
|5-10 MHz transducer: CL3.8 5-2.5 MHz 60 mm curved linear array||E.I. Medical Imaging, Loveland, CO||290420|
|3 ml syringes||Covidien||1.18E+09|
|22 G needles||Covidien||8.88E+09|
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