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.

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.
<ol>
	<li>Prior to examination, immobilize animals using appropriate sedation or anesthesia protocols to provide relaxation and allow for appropriate positioning. One handler restrains the deer while a second person administers the sedation to the animal. 
	Note: The authors have found the following combination to provide effective sedation in this species: butorphanol (0.45 mg/kg), azaperone (0.035 mg/kg), and medetomidine (0.04 mg/kg), administered intramuscularly in the gluteal muscles.</li>
	<li>Once the doe has reached a plane of sedation that allows for compliant handling, position her in either dorsal or lateral recumbency on an examination table or within a V-trough. Elevate the head, neck, and thorax slightly (approximately 20-30&#176;) to reduce the risk of regurgitation and aspiration of rumenal contents and to facilitate proper ventilation during the procedure. 
	Note: In most cervids or other ruminant species, abdominal ultrasound is performed in standing animals; however, the small size and behavioral considerations of this species requires heavy sedation for examination and sternal positioning is therefore more difficult and may preclude effective imaging technique. Positioning the animal midway between sternal and lateral recumbency may be ideal for reducing the risk of regurgitation and hypoventilation and should be considered if possible. In any recumbent position, it is critical to carefully monitor the animals&#39; respirations and immediately stop the examination and place the animal in sternal recumbency if regurgitation or hypoventilation is noted.</li>
	<li>If necessary, clip hair from the area of the abdomen to be examined in order to achieve adequate contact between the footprint of the transducer and the skin. 
	Note: In early gestation, evidence of pregnancy can be detected in the caudal abdomen near the mammary glands. This region of the muntjac is typically hairless, and therefore shaving may not be indicated at this time. As the fetus grows and the uterus distends to occupy a more cranial position in the abdomen, clipping of the hair will be necessary prior to imaging. Care should be taken to avoid causing any mechanical trauma to the skin, including lacerations from razor blades or thermal injury from electrical clippers.</li>
	<li>Position the ultrasound unit in a location that allows the technician to easily visualize the monitor while conducting the examination with his or her dominant hand. Input animal identification information (name, identification number, etc.) into the system to automatically label saved images and video loops.</li>
</ol>
2. Transabdominal Reproductive Ultrasound Examination
<ol>
	<li>Apply a generous amount of acoustic coupling gel to the caudal abdomen.</li>
	<li>Place a 5-10 MHz curved linear transducer on the skin of the caudal abdomen, immediately cranial to the pubis, and move it slowly in a cranial direction to evaluate the structures within the pelvic cavity. Scan the left and right quadrants of the caudal abdomen to assess both horns of the muntjac&#39;s bicornuate uterus. 
	Note: Comprehensive abdominal ultrasound involves assessment of all abdominal viscera, including the liver, kidneys, spleen, gastrointestinal tract, urinary bladder, and reproductive tract. For the purposes of this protocol, only examination of pregnancy will be discussed in detail; however, the normal appearance of other caudal abdominal organs should be recognized as these structures are likely to be encountered during the course of the study. Organs most commonly seen in this location include segments of the gastrointestinal tract, the urinary bladder, and the reproductive tract. The large and small intestines are identified by the characteristically layered appearance of the intestinal wall. The lumen may or may not be pronounced depending on the type and amount of contents, which may include fluid, gas, mucous, or food particles. The urinary bladder may be difficult to identify unless distended with urine, and the layers of the bladder wall are significantly less distinct than those of the intestines. Apart from the uterus, the ovaries may also be found on ultrasound of the reproductive tract of the female muntjac. The ovaries will appear as ovoid structures near the caudal pole of the kidneys, and their appearance may change over the course of the estrous cycle. The presence of multiple mature, fluid-filled follicles within the cortex is suggestive of estrus. 
	Note: By rotating and/or &#34;fanning&#34; the scanhead of the transducer, structures can be evaluated in transverse or sagittal planes.
	<ol>
		<li>If needed to differentiate a gravid uterus from the urinary bladder, empty the bladder manually by applying gentle pressure to the caudal abdomen or by placing a urinary catheter and repeating the scan. 
		Note: Manual expression of the bladder should only be attempted when the animal is in a sternal or upright position, which facilitates a more natural orientation of the abdominal viscera and reduces the risk of trauma to other structures. The uterus may also be differentiated from the urinary bladder by assessing the thickness of the organ&#39;s wall. The typical thickness of a moderately distended bladder is approximately 1.4 mm24, while the authors have found that the more muscular wall of the gravid uterus in the muntjac generally measures between 7-8 mm. 
		Note: Early pregnancy is difficult to diagnose definitively due to a lack of identifiable fetal structures, and in fact the first indication of pregnancy may simply be a small fluid pocket in the caudal abdomen. If seen at this time, the developing embryo will appear as a hyperechoic, ovoid to amorphous structure within the uterus (Figure 1).</li>
	</ol>
	</li>
	<li>Make adjustments to the depth, frequency, and gain as necessary to improve contrast and image detail. 
	Note: higher frequencies are associated with shorter wavelengths, which are necessary for improved image resolution. Most transducers are capable of operating within a range of frequencies (for example, 6-10 MHz)24. Frequency can be adjusted using the scanner controls on the ultrasound unit, and increasing frequency will result in a clearer image. Increasing frequency concurrently decreases the depth of sound wave penetration; therefore, reducing the frequency may be necessary to visualize deeper structures, but resolution may become compromised24. For this reason it is important to select the highest frequency possible to reach the desired tissue depth. Adjustments to the gain controls will alter the amplification of sound waves returning to the transducer from the tissues and affect the degree of contrast in the image24. The gain should be set to a point where optimal contrast between hyperechoic and hypoechoic tissues is achieved, and these settings may need to be adjusted depending on the structures that are being imaged.</li>
</ol>
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&#39; 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&#39; muntjac as early as 35 days post-observed mating.
<ol>
	<li>To measure SCRL, locate the uterus on the monitor and make fine adjustments to the position of the transducer until a sagittal view of the entire fetus is obtained (Figures 2 and 3). Freeze the image and measure the distance between the most dorsal aspect of the skull and the base of the sacrum (Figures 2 and 3).</li>
	<li>To measure CD, obtain a sagittal view of the fetus (the same image may be used to measure SCRL) and measure the distance between the dorsal and ventral aspects of the thorax (Figures 2 and 3).</li>
	<li>To measure HL, using the same sagittal view of the fetus as described for the SCRL and CD measurements, measure the distance from the occipital crest to the base of nose (Figures 2 and 3).</li>
	<li>To calculate fetal heart rate, identify the heart within the thoracic cavity of the fetus by visualizing its rhythmical contractions. Calculate the heart rate by counting the number of contractions (heart beats) that occur within a 1 min period of time. 
	Note: If the ultrasound unit being used has Doppler capabilities, this feature may be employed to further assist in distinguishing the heart from other nonspecific fetal movements.</li>
	<li>Verify that all saved images and video loops include all necessary identifying information, including animal name or number, date, measurements, and appropriate structure labels.</li>
</ol>
4. Procedural Conclusions and Recovery
<ol>
	<li>Remove all residual ultrasound contact gel from the animal&#39;s abdomen using a soft cloth or paper towels. Clean the gel from the transducer prior to storage.</li>
	<li>If using the sedation protocol described in step 1.1, administer atipamezole subcutaneously at a dose of 0.25 mg/kg to reverse the effects of medetomidine. If using a different sedation protocol in which an antagonist is available, administer this agent to the animal according to published dosages at the conclusion of the procedure to speed recovery.</li>
	<li>Place the muntjac on a soft bedded surface in sternal recumbency, with the head slightly elevated to reduce the risk of regurgitation and aspiration of rumenal contents. Monitor the animal closely until fully recovered and able to ambulate around the enclosure without assistance.</li>
</ol>

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+ultrasonic+scanning+in+the+diagnosis+of+pregnancy+and+the+estimation+of+gestational+age+in+cattle.">Real-time ultrasonic scanning in the diagnosis of pregnancy and the estimation of gestational age in cattle.</a> Vet Rec. 117 (1), 5-8 (1985).</li><li>Sergeev, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+ultrasound+imaging+for+predicting+ovine+fetal+age.">Real-time ultrasound imaging for predicting ovine fetal age.</a> Theriogenology. 34 (3), 593-601 (1990).</li><li>Aiumlamai, S., Fredriksson, G., Nilsfors, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+ultrasonography+for+determining+the+gestational+age+of+ewes.">Real-time ultrasonography for determining the gestational age of ewes.</a> Vet. Rec. 131 (24), 560-562 (1992).</li><li>Lee, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasonic+measurement+of+fetal+parameters+for+estimation+of+gestational+age+in+Korean+black+goats.">Ultrasonic measurement of fetal parameters for estimation of gestational age in Korean black goats.</a> J. Vet. Med. Sci. 67 (5), 497-502 (2005).</li><li>Renaudin, C. D., Gillis, C. L., Tarantal, A. F., Coleman, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+equine+fetal+growth+from+day+100+of+gestation+to+parturition+by+ultrasonography.">Evaluation of equine fetal growth from day 100 of gestation to parturition by ultrasonography.</a> J. Reprod. Fertil. Suppl. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+pregnancy+and+prediction+of+fetal+age+in+red+deer+by+real-time+ultrasonic+scanning.">Diagnosis of pregnancy and prediction of fetal age in red deer by real-time ultrasonic scanning.</a> Vet. Rec. 124 (15), 395-397 (1989).</li><li>Bingham, C. M., Wilson, P. R., Davies, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+ultrasonography+for+pregnancy+diagnosis+and+estimation+of+fetal+age+in+farmed+red+deer.">Real-time ultrasonography for pregnancy diagnosis and estimation of fetal age in farmed red deer.</a> Vet. Rec. 126 (5), 102-106 (1990).</li><li>Wilson, P. R., Bingham, C. 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G., Furlong, M., Harris, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproductive+strategies+and+the+influence+of+date+of+birth+on+growth+and+sexual+development+of+an+aseasonally-breeding+ungulate:+Reeves'+muntjac+(Muntiacus+reevesi).">Reproductive strategies and the influence of date of birth on growth and sexual development of an aseasonally-breeding ungulate: Reeves' muntjac (Muntiacus reevesi).</a> J. Zool. 241 (3), 551-570 (1996).</li><li>Liming, S., Yingying, Y., Xingsheng, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+cytogenetic+studies+on+the+red+muntjac,+Chinese+muntjac,+and+their+F1+hybrids.">Comparative cytogenetic studies on the red muntjac, Chinese muntjac, and their F1 hybrids.</a> Cytogenet. Cell Genet. 26 (1), 22-27 (1980).</li><li>Nyland, T. G., Mattoon, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chapter 2. Small Animal Diagnostic Ultrasound. , 19-29 (2002).</li><li>Huang, F., Cockrell, D. C., Stephenson, T. R., Noyes, J. H., Sasser, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+serum+pregnancy+test+with+a+specific+radioimmunoassay+for+moose+and+elk+pregnancy-specific+protein+B.">A serum pregnancy test with a specific radioimmunoassay for moose and elk pregnancy-specific protein B.</a> J. Wildl. Manag. 64, 492-499 (2000).</li></ol>

The authors have nothing to disclose.

This protocol describes the application of transabdominal ultrasonography for pregnancy diagnosis in Reeves&#39; 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&#39; muntjac. The value of this technique, however, is highly dependent upon the specific capabilities of the animals&#39; 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&#39; 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&#39; 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.

Ultrasonographic examination is a well-described method of pregnancy diagnosis in cattle1, sheep2,3, goats4, horses5, companion animals6,7,&#160;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&#39; 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&#39; 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&#160;and may also be useful for clinical purposes in captive breeding operations.

<table><tbody><tr><td>Butorphanol tartrate (Torbugesic) (0.45 mg/kg)</td><td>Fort Dodge Animal Health, Fort Dodge, Iowa</td><td>NADA 135-780</td><td></td></tr><tr><td>Azaperone tartrate (0.035 mg/kg)</td><td>ZooPharm</td><td></td><td></td></tr><tr><td>Medetomidine HCl (0.04 mg/kg)</td><td>ZooPharm</td><td></td><td></td></tr><tr><td>Ultrasound contact gel</td><td>Medline, Mundelein, Illinois</td><td>MDS092005</td><td></td></tr><tr><td>Atipamezole hydrochloride (Antisedan) (0.25 mg/kg)</td><td>Orion Corporation, Espoo, Finland</td><td>NADA 141-033</td><td></td></tr><tr><td>V-trough (optional)</td><td>N/A</td><td>N/A</td><td>Animal may be supported in dorsal recumbency on a standard examination table or other surface without the use of a V-trough</td></tr><tr><td>Electrical clippers or razor blades for hair removal</td><td>Oster, McMinnville, Tennessee</td><td>78005-140</td><td></td></tr><tr><td>Ultrasound system: Ibex Pro portable ultrasound</td><td>E.I. Medical Imaging, Loveland, CO</td><td>Ibex Pro</td><td></td></tr><tr><td>5-10 MHz transducer: CL3.8 5-2.5 MHz 60 mm curved linear array</td><td>E.I. Medical Imaging, Loveland, CO</td><td>290420</td><td></td></tr><tr><td>3 ml syringes</td><td>Covidien</td><td>1.18E+09</td><td></td></tr><tr><td>22 G needles</td><td>Covidien</td><td>8.88E+09</td><td></td></tr></tbody></table>

transabdominal ultrasound for pregnancy diagnosis in reeves' muntjac deer

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.

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.
<img alt="Ultrasound scan, embryo measurement, uterus, animal research, reproductive study, gestation analysis." fo:content-width="5in" fo:src="/files/ftp_upload/50855/50855fig1highres.jpg" src="/files/ftp_upload/50855/50855fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
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.
<img alt="Ultrasound image with fetal measurements diagram; prenatal development analysis." fo:content-width="5in" fo:src="/files/ftp_upload/50855/50855fig2highres.jpg" src="/files/ftp_upload/50855/50855fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
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).
<img alt="Ultrasound image showing fetal measurements; CRL, CD, HL in animal research lab study." fo:content-width="5in" fo:src="/files/ftp_upload/50855/50855fig3highres.jpg" src="/files/ftp_upload/50855/50855fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
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).

Watch this Scientific Journal Video about Transabdominal Ultrasound for Pregnancy Diagnosis in Reeves' Muntjac Deer at JoVE.com

Transabdominal Ultrasound for Pregnancy Diagnosis in Reeves' Muntjac Deer

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.

Reeves' muntjac deer (Muntiacus reevesi) are a small cervid species native to southeast Asia, and are currently being investigated as a potential model of ...

transabdominal-ultrasound-for-pregnancy-diagnosis-reeves-muntjac

Nanyang Technological University

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JoVE Journal

Medicine

10.3K Views.  Colorado State University. 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.

Video: Transabdominal Ultrasound for Pregnancy Diagnosis in Reeves' Muntjac Deer

Murine Fetal Echocardiography

Transgenic mice displaying abnormalities in cardiac development and function represent a powerful tool for the understanding the molecular mechanisms underlying both normal cardiovascular function and the pathophysiological basis of human cardiovascular disease. Fetal and perinatal death is a common feature when studying genetic alterations affecting cardiac development 1-3. In order to study the role of genetic or pharmacologic alterations in the early development of cardiac function, ultrasound imaging of the live fetus has become an important tool for early recognition of abnormalities and longitudinal follow-up. Noninvasive ultrasound imaging is an ideal method for detecting and studying congenital malformations and the impact on cardiac function prior to death 4. It allows early recognition of abnormalities in the living fetus and the progression of disease can be followed in utero with longitudinal studies 5,6. Until recently, imaging of fetal mouse hearts frequently involved invasive methods. The fetus had to be sacrificed to perform magnetic resonance microscopy and electron microscopy or surgically delivered for transillumination microscopy. An application of high-frequency probes with conventional 2-D and pulsed-wave Doppler imaging has been shown to provide measurements of cardiac contraction and heart rates during embryonic development with databases of normal developmental changes now available 6-10. M-mode imaging further provides important functional data, although, the proper imaging planes are often difficult to obtain. High-frequency ultrasound imaging of the fetus has improved 2-D resolution and can provide excellent information on the early development of cardiac structures 11.

Fetal and perinatal death is a common feature when studying genetic alterations affecting cardiac development. High-frequency ultrasound imaging has improved 2-D resolution and can provide excellent information on early cardiac development and is an ideal method to detect the impact on cardiac structure and function prior to death.

Transgenic mice displaying abnormalities in  cardiac development and function represent a powerful tool for the understanding  the molecular mechanisms ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

Developmental Biology

Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ultrasound and fetal echocardiography. Increasingly, these imaging modalities are being employed clinically to examine cardiac function and assess wellbeing in utero, thereby guiding timing of birth decisions. Here, we used a rabbit model of IUGR that allows analysis of cardiac function in a clinically relevant way. Using isoflurane induced anesthesia, IUGR is surgically created at gestational age day 25 by performing a laparotomy, exposing the bicornuate uterus and then ligating 40-50% of uteroplacental vessels supplying each gestational sac in a single uterine horn. The other horn in the rabbit bicornuate uterus serves as internal control fetuses. Then, after recovery at gestational age day 30 (full term), the same rabbit undergoes examination of fetal cardiac function. Anesthesia is induced with ketamine and xylazine intramuscularly, then maintained by a continuous intravenous infusion of ketamine and xylazine to minimize iatrogenic effects on fetal cardiac function. A repeat laparotomy is performed to expose each gestational sac and a microultrasound examination (VisualSonics VEVO 2100) of fetal cardiac function is performed. Placental insufficiency is evident by a raised pulsatility index or an absent or reversed end diastolic flow of the umbilical artery Doppler waveform. The ductus venosus and middle cerebral artery Doppler is then examined. Fetal echocardiography is performed by recording B mode, M mode and flow velocity waveforms in lateral and apical views. Offline calculations determine standard M-mode cardiac variables, tricuspid and mitral annular plane systolic excursion, speckle tracking and strain analysis, modified myocardial performance index and vascular flow velocity waveforms of interest. This small animal model of IUGR therefore affords examination of in utero cardiac function that is consistent with current clinical practice and is therefore useful in a translational research setting.

We describe examination of fetal cardiac function with contemporary functional fetal echocardiography and fetoplacental Doppler ultrasound using the VisualSonics VEVO 2100 microultrasound in a surgically induced model of intrauterine fetal growth restriction in a rabbit.

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ...

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Ultrasound imaging is a widespread method used to detect organ anomalies and tumors in human and animal tissues. The method is non-invasive, harmless, and painless, and the application is easy, fast, and can be done anywhere, even with mobile devices. During pregnancy, ultrasound imaging is standardly used to closely monitor fetal development. The technique is important to assess intrauterine growth restriction (IUGR), a pregnancy complication with short- and long-term health consequences for both the mother and fetus. Understanding the process of IUGR is indispensable for developing effective therapeutic strategies. 
The ultrasound system used in this manuscript is an ultrasound device produced for the analysis of small animals and can be used in various research fields, including pregnancy research. Here we describe the usage of the system for in vivo analysis of fetuses from natural killer (NK) cell/mast cell (MC)-deficient mothers that give birth to growth-restricted pups. The protocol includes preparation of the system, handling of the mice before and during measurements, and the usage of the B-mode, color doppler mode, and pulse-wave doppler mode. Fetal size, placental size, and blood supply to the fetus were analyzed. We found reduced implantation sizes and smaller placentas in NK/MC-deficient mice from mid-gestation onwards. In addition, MC/NK-deficiency was associated with absent and reversed end diastolic flow in the fetal Arteria umbilicalis(UmA) and an elevated resistance index. The methods described in the protocol can easily be used for related and non-related research topics.

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Here we describe the technique of high frequency ultrasound for in vivo analysis of fetuses in mice. This method allows the follow-up of fetuses and the analysis of placental parameters as well as maternal and fetal blood flow throughout pregnancy.

Ultrasound imaging is a widespread method used to detect organ anomalies and tumors in human and animal tissues. The method is non-invasive, harmless, and ...

Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice

Uterine cancers can be studied in mice due to the ease of handling and genetic manipulation in these models. However, these studies are often limited to assessing pathology post-mortem in animals euthanized at multiple time points in different cohorts, which increases the number of mice needed for a study. Imaging mice in longitudinal studies can track the progression of disease in individual animals, reducing the number of mice needed. Advances in ultrasound technology have allowed for the detection of micrometer-level changes in tissues. Ultrasound has been used to study follicle maturation in ovaries and xenograft growth but has not been applied to morphological changes in the mouse uterus. This protocol examines the juxtaposition of pathology with in vivo imaging comparisons in an induced endometrial cancer mouse model. The features observed by ultrasound were consistent with the degree of change seen by gross pathology and histology. Ultrasound was found to be highly predictive of the observed pathology, supporting the incorporation of ultrasonography into longitudinal studies of uterine diseases such as cancer in mice.

Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice

This protocol describes a method for monitoring the progression of morphological changes over time in the uterus in an inducible mouse model of endometrial cancer using ultrasound imaging with correlation to gross and histological changes.

Uterine cancers can be studied in mice due to the ease of handling and genetic manipulation in these models. However, these studies are often limited to ...

Cancer Research

Is My Mouse Pregnant? High-Frequency Ultrasound Assessment

The mouse is the mammalian animal model of choice for many human diseases and biological processes. Developmental biology often requires staged-pregnant mice to determine evolving processes at various timepoints. Moreover, optimal and efficient breeding of model mice requires an assessment of timed pregnancies. Most commonly, mice are mated overnight, and the presence of a vaginal plug is determined; however, the positive predictive value of this technique is suboptimal, and one needs to wait to know if the mouse is truly pregnant. High-resolution ultrasound biomicroscopy is an effective and efficient tool for imaging: 1) Whether a mouse is pregnant; 2) What gestational stage the mouse has reached; and 3) Whether there are intrauterine losses. In addition to the embryos and fetuses, the investigator must also recognize common artifacts in the abdominal cavity so as not to mistake these for a gravid uterus. This article provides a protocol for imaging along with illustrative examples.

High-resolution ultrasound can help streamline experiments requiring timed-pregnant mice by determining the state of pregnancy, gestational age, and pregnancy losses. Presented here is a protocol to illustrate methods to assess mouse pregnancies as well as potential pitfalls (image artifacts) that may mimic pregnancy.

The mouse is the mammalian animal model of choice for many human diseases and biological processes. Developmental biology often requires staged-pregnant ...

Murine Neural Plate Targeting by In Utero Nano-Injection (NEPTUNE) at Embryonic Day 7.5

Manipulating gene expression in the developing mouse brain in utero holds great potential for functional genetics studies. However, it has previously largely been restricted to the manipulation of embryonic stages post-neurulation. A protocol was developed to inject the amniotic cavity at embryonic day (E)7.5 and deliver lentivirus, encoding cDNA or shRNA, targeting &gt;95% of the neural plate and neural crest cells, contributing to the future brain, spinal cord, and peripheral nervous system. This protocol describes the steps necessary to achieve successful transduction, including grinding of the glass capillary needles, pregnancy verification, developmental staging using ultrasound imaging, and optimal injection volumes matched to embryonic stages. 
Following this protocol, it is possible to achieve transduction of &gt;95% of the developing brain with high-titer lentivirus and thus perform whole-brain genetic manipulation. In contrast, it is possible to achieve mosaic transduction using lower viral titers, allowing for genetic screening or lineage tracing. Injection at E7.5 also targets ectoderm and neural crest contributing to distinct compartments of the eye, tongue, and peripheral nervous system. This technique thus offers the possibility to manipulate gene expression in mouse neural-plate- and ectoderm-derived tissues from preneurulation stages, with the benefit of reducing the number of mice used in experiments.

Murine Neural Plate Targeting by In Utero Nano-Injection NEPTUNE at Embryonic Day 7.5

In this protocol, we describe how to inject the mouse amniotic cavity at E7.5 with lentivirus, leading to uniform transduction of the entire neural plate, with minimal detrimental effects on survival or embryonic development.

Manipulating gene expression in the developing mouse brain in utero holds great potential for functional genetics studies. However, it has previously ...

Ultrasonography in Experimental Reproductive Investigations on Rats

With the development of assisted reproductive technology and the ethical limitations of research on humans, rat animal models have been widely used in reproductive medicine. In the past, the study of reproductive system development in rodents has been based on one-time histological examination of excised tissues. Recently, with the development of high-resolution transabdominal ultrasound, high-quality sonography can now be performed to evaluate the reproductive organs of rats, allowing a new method for studying the reproductive system. Images were obtained using a high-resolution ultrasonographic system. Gynecological ultrasonography was performed on 28 eight-week-old non-pregnant rats and 5 pregnant Sprague-Dawley rats. We describe how to recognize organs of the reproductive system and associated structures in typical views during different phases of the estrus cycle. Color flow Doppler was used to measure uterine artery blood flow and evaluate uterine blood flow pattern changes during different stages of pregnancy. We have demonstrated that ultrasound exploration is a useful method for evaluating changes in internal reproductive organs. Its use raises the possibility of conducting additional experiments, including medical or surgical procedures, and provides the ability to monitor sonographic changes to internal organs without sacrificing animals.

This manuscript describes the utility of ultrasound performed on female rats to design experimental models for reproductive and gynecological investigation. A step-by-step explanation of how to perform ultrasonographic evaluation is shown.

With the development of assisted reproductive technology and the ethical limitations of research on humans, rat animal models have been widely used in ...

Milk Collection Methods for Mice and Reeves' Muntjac Deer

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native host. Milk collection from animals allows scientists the opportunity to study many aspects of reproduction including vertical transmission, passive immunity, mammary gland biology, and lactation. Obtaining adequate volumes of milk for these studies is a challenging task, especially from small animal models. Here we illustrate an inexpensive and facile method for milk collection in mice and Reeves&#8217; muntjac deer that does not require specialized equipment or extensive training. This particular method requires two researchers: one to express the milk and to stabilize the animal, and one to collect the milk in an appropriate container from either a Muntjac or mouse model. The mouse model also requires the use of a P-200 pipetman and corresponding pipette tips. While this method is low cost and relatively easy to perform, researchers should be advised that anesthetizing the animal is required for optimal milk collection.

Milk collection from animal models facilitates various research avenues: understanding passive immunity, identifying pathogens responsible for vertical transmission and, through the use of transgenic mice, even commercial production of proteins found in human breast milk. Here we illustrate a simple method for milk collection in mice and Reeves&#8217; muntjac deer.

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native ...

Biology

Reproductive Techniques for Ovarian Monitoring and Control in Amphibians

Ovarian control and monitoring in amphibians require a multi-faceted approach. There are several applications that can successfully induce reproductive behaviors and the acquisition of gametes and embryos for physiological or molecular research. Amphibians contribute to one-quarter to one-third of vertebrate research, and of interest in this context is their contribution to the scientific community&#39;s knowledge of reproductive processes and embryological development. However, most of this knowledge is derived from a small number of species. In recent times, the decimation of amphibians across the globe has required increasing intervention by conservationists. The captive recovery and assurance colonies that continue to emerge in response to the extinction risk make existing research and clinical applications invaluable to the survival and reproduction of amphibians held under human care. The success of any captive population is founded on its health and reproduction and the ability to develop viable offspring that carry forward the most diverse genetic representation of their species. For researchers and veterinarians, the ability to monitor and control ovarian development and health is, therefore, imperative. The focus of this article is to highlight the different assisted reproductive techniques that can be used to monitor and, where appropriate or necessary, control ovarian function in amphibians. Ideally, any reproductive and health issues should be reduced through proper captive husbandry, but, as with any animal, issues of health and reproductive pathologies are inevitable. Non-invasive techniques include behavioral assessments, visual inspection and palpation and morphometric measurements for the calculation of body condition indices and ultrasound. Invasive techniques include hormonal injections, blood sampling, and surgery. Ovarian control can be exercised in a number of ways depending on the application required and species of interest.

The study of amphibian biology provides valuable information on the reproductive, physiological, embryological and developmental processes that drive organisms of many taxonomic groups. Here, we present a comprehensive guide on different methodologies that can be used to study ovarian control and monitoring in amphibians.

Ovarian control and monitoring in amphibians require a multi-faceted approach. There are several applications that can successfully induce reproductive ...

Transabdominal Ultrasound for Pregnancy Diagnosis in Reeves' Muntjac Deer

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Murine Fetal Echocardiography

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice

Is My Mouse Pregnant? High-Frequency Ultrasound Assessment

Murine Neural Plate Targeting by In Utero Nano-Injection (NEPTUNE) at Embryonic Day 7.5

Ultrasonography in Experimental Reproductive Investigations on Rats

Milk Collection Methods for Mice and Reeves' Muntjac Deer

Reproductive Techniques for Ovarian Monitoring and Control in Amphibians