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>

<ol>
	<li>White, I. R., Russel, A. J., Wright, I. A., Whyte, T. 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. (56), 651-660 (2000).</li><li>Lopate, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+gestational+age+and+assessment+of+canine+fetal+maturation+using+radiology+and+ultrasonography:+a+review.">Estimation of gestational age and assessment of canine fetal maturation using radiology and ultrasonography: a review.</a> Theriogenology. 70 (3), 397-402 (2008).</li><li>Gatel, L., Rosset, E., Chalvet-Monfray, K., Buff, S., Rault, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationships+between+fetal+biometry,+maternal+factors+and+birth+weight+of+purebred+domestic+cat+kittens.">Relationships between fetal biometry, maternal factors and birth weight of purebred domestic cat kittens.</a> Theriogenology. 76 (9), 1716-1722 (2011).</li><li>Mulley, R. C., English, A. W., Rawlinson, R. J., Chapple, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pregnancy+diagnosis+of+fallow+deer+by+ultrasonography.">Pregnancy diagnosis of fallow deer by ultrasonography.</a> Aust. Vet. J. 64 (8), 257-258 (1987).</li><li>Willard, S. T., Sasser, R. G., Jaques, J. T., White, D. R., Neuendorff, D. A., Randel, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+pregnancy+detection+and+the+hormonal+characterization+of+embryonic-fetal+mortality+in+fallow+deer+(Dama+dama).">Early pregnancy detection and the hormonal characterization of embryonic-fetal mortality in fallow deer (Dama dama).</a> Theriogenology. 49 (4), 861-869 (1998).</li><li>Willard, S. T., Petty, S. J., Sasser, R. G., White, D. L., Randel, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pregnancy+detection+and+the+effects+of+age,+body+weight,+and+previous+reproductive+performance+on+pregnancy+status+and+weaning+rates+of+farmed+fallow+deer+(Dama+dama).">Pregnancy detection and the effects of age, body weight, and previous reproductive performance on pregnancy status and weaning rates of farmed fallow deer (Dama dama).</a> J. Anim. Sci. 77 (1), 32-38 (1999).</li><li>Lenz, M. F., English, A. W., Dradjat, A. <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+foetal+ageing+in+fallow+deer.">Real-time ultrasonography for pregnancy diagnosis and foetal ageing in fallow deer.</a> Aust. Vet. J. 70 (10), 373-375 (1993).</li><li>White, I. R., McKelvey, W. A., Busby, S., Sneddon, A., Hamilton, W. 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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+of+pregnancy+diagnosis+and+prediction+of+calving+date+in+red+deer+using+real-time+ultrasound+scanning.">Accuracy of pregnancy diagnosis and prediction of calving date in red deer using real-time ultrasound scanning.</a> Vet. Rec. 126 (6), 133-135 (1990).</li><li>Revol, B., Wilson, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasonography+of+the+reproductive+tract+and+early+pregnancy+in+red+deer.">Ultrasonography of the reproductive tract and early pregnancy in red deer.</a> Vet. Rec. 128 (10), 229-233 (1991).</li><li>Revol, B., Wilson, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foetal+ageing+in+farmed+red+deer+using+real-time+ultrasonography.">Foetal ageing in farmed red deer using real-time ultrasonography.</a> Anim. Reprod. Sci. 25 (3), 241-253 (1991).</li><li>Yanagawa, 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=Fetal+age+estimation+of+Hokkaido+sika+deer+(Cervus+nippon+yesoensis)+using+ultrasonography+during+early+pregnancy.">Fetal age estimation of Hokkaido sika deer (Cervus nippon yesoensis) using ultrasonography during early pregnancy.</a> J. Reprod. Dev. 55 (2), 143-148 (2009).</li><li>Savela, H., Vahtiala, S., Lindeberg, H., Dahl, R. E., Beckers, J. F., Saarela, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+accuracy+of+ultrasonography,+progesterone,+and+pregnancy-associated+glycoprotein+tests+for+pregnancy+diagnosis+in+semidomesticated+reindeer.">Comparison of accuracy of ultrasonography, progesterone, and pregnancy-associated glycoprotein tests for pregnancy diagnosis in semidomesticated reindeer.</a> Theriogenology. 72 (9), 1229-1236 (2009).</li><li>Vahtiala, S., Sakkinen, H., Dahl, E., Beckers, J. F., Ropstad, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasonography+in+early+pregnancy+diagnosis+and+measurements+of+fetal+size+in+reindeer+(Rangifer+tarandus+tarandus).">Ultrasonography in early pregnancy diagnosis and measurements of fetal size in reindeer (Rangifer tarandus tarandus).</a> Theriogenology. 61 (4), 785-795 (2004).</li><li>Chapman, N. G., 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=Evidence+that+the+seasonal+antler+cycle+of+adult+Reeves'+muntjac+(Muntiacus+reevesi)+is+not+associated+with+reproductive+quiescence.">Evidence that the seasonal antler cycle of adult Reeves' muntjac (Muntiacus reevesi) is not associated with reproductive quiescence.</a> J. Reprod. Fertil. 92 (2), 361-369 (1991).</li><li>Chapman, D. I., Chapman, N. G., Dansie, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+periods+of+conception+and+parturition+in+feral+Reeves'+muntjac+(Muntiacus+reevesi)+in+southern+England,+based+upon+age+of+juvenile+animals.">The periods of conception and parturition in feral Reeves' muntjac (Muntiacus reevesi) in southern England, based upon age of juvenile animals.</a> J. Zool. 204 (4), 575-578 (1984).</li><li>Chapman, N. 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...

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 border="0" cellpadding="0" cellspacing="0" width="547">
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	<tbody>
		<tr height="61">
			<td height="61" style="height:61px;width:187px;">Butorphanol tartrate (Torbugesic) (0.45 mg/kg)</td>
			<td style="width:127px;">Fort Dodge Animal Health, Fort Dodge, Iowa</td>
			<td style="width:64px;">NADA 135-780</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:187px;">Azaperone tartrate (0.035 mg/kg)</td>
			<td style="width:127px;">ZooPharm</td>
			<td style="width:64px;"></td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:187px;">Medetomidine HCl (0.04 mg/kg)</td>
			<td style="width:127px;">ZooPharm</td>
			<td style="width:64px;"></td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:187px;">Ultrasound contact gel</td>
			<td style="width:127px;">Medline, Mundelein, Illinois</td>
			<td style="width:64px;">MDS092005</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:187px;">Atipamezole hydrochloride (Antisedan) (0.25 mg/kg)</td>
			<td style="width:127px;">Orion Corporation, Espoo, Finland</td>
			<td style="width:64px;">NADA 141-033</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
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			<td height="21" style="height:21px;">Table 2 Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:187px;">Name of Equipment</td>
			<td style="width:127px;">Company</td>
			<td style="width:64px;">Catalog Number</td>
			<td style="width:171px;">Comments</td>
		</tr>
		<tr height="201">
			<td height="201" style="height:201px;width:187px;">V-trough (optional)</td>
			<td style="width:127px;">N/A</td>
			<td style="width:64px;">N/A</td>
			<td style="width:171px;">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 height="61">
			<td height="61" style="height:61px;width:187px;">Electrical clippers or razor blades for hair removal</td>
			<td style="width:127px;">Oster, McMinnville, Tennessee</td>
			<td style="width:64px;">78005-140</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:187px;">Ultrasound system: Ibex Pro portable ultrasound</td>
			<td style="width:127px;">E.I. Medical Imaging, Loveland, CO</td>
			<td style="width:64px;">Ibex Pro</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:187px;">5-10 MHz transducer: CL3.8 5-2.5 MHz 60 mm curved linear array</td>
			<td style="width:127px;">E.I. Medical Imaging, Loveland, CO</td>
			<td align="right" style="width:64px;">290420</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:187px;">3 ml syringes</td>
			<td style="width:127px;">Covidien</td>
			<td align="right" style="width:64px;">1.18E+09</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:187px;">22 G needles</td>
			<td style="width:127px;">Covidien</td>
			<td align="right" style="width:64px;">8.88E+09</td>
			<td style="width:171px;"></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&#39;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 ...

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&#039; 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.

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 ...

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

Research

JoVE Journal

Medicine

10.2K ビュー.  Colorado State University. 腹腔内超音波は、リーブスのムンジャク鹿の生殖状態を評価するための効果的で非侵襲的な手段として記述されている。これらの方法は、妊娠初期の診断を達成し、胎児の生存率を評価するために使用することができる。この技術の将来の応用には、妊娠年齢の推定と胎児の発達に及ぼす母親の病気の影響が含まれる。

ビデオ: Transabdominal Ultrasound for Pregnancy Diagnosis in Reeves' Muntjac Deer

FISH for Pre-implantation Genetic Diagnosis

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a cohort generated by assisted reproduction technology (ART). This selection may be required because of familial monogenic disease (e.g. cystic fibrosis), or because one partner carries a chromosome rearrangement (e.g. a two-way reciprocal translocation). PGD is available for couples who have had previous affected children, and/or in the case of chromosome rearrangements, recurrent miscarriages, or infertility. Oocytes aspirated following ovarian stimulation are fertilized by in vitro immersion in semen (IVF) or by intracytoplasmic injection of an individual spermatozoon (ICSI). Pre-implantation cleavage-stage embryos are biopsied, usually by the removal of a single cell on day 3 post-fertilization, and the biopsied cell is tested to establish the genetic status of the embryo. Fluorescence in situ hybridization (FISH) on the fixed nuclei of biopsied cells with target-specific DNA probes is the technique of choice to detect chromosome imbalance associated with chromosome rearrangements, and to select female embryos in families with X-linked disease for which there is no mutation-specific test. FISH has also been used to screen embryos for spontaneous chromosome aneuploidy (also known as PGS or PGD-AS) in order to try and improve the efficiency of assisted reproduction; however, the predictive value of this test using the spreading and FISH technique described here is likely to be unacceptably low in most people's hands and it is not recommended for routine clinical use. We describe the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to PGD in a clinical setting.

This article describes the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to pre-implantation genetic diagnosis (PGD) in a clinical setting.

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a ...

Hydra, a Computer-Based Platform for Aiding Clinicians in Cardiovascular Analysis and Diagnosis

Cardiovascular diseases (CVDs) are the leading cause of death throughout the world. The total risk of developing CVD is determined by the combined effect of different cardiovascular risk factors (e.g., diabetes, raised blood pressure, unhealthy diet, tobacco use, stress, etc.) that commonly coexist and act multiplicatively. Most CVDs can be prevented by an early identification of the highest risk factors and an appropriate treatment. The stratification of cardiovascular risk factors involves a wide range of parameters and tests that specialists use in their clinical practice. In addition to cardiovascular (CV) risk stratification, ambulatory blood pressure monitoring (ABPM) also provides relevant information for diagnostic and treatment purposes. This work presents a list of protocols based on the Hydra platform, a web-based system for clinical decision support which incorporates a set of functionalities and services that are required for complete cardiovascular analysis, risk assessment, early diagnosis, treatment and monitoring of patients over time. The program includes tools for inputting and managing comprehensive patient data, organized into different checkups to track the evolution over time. It also has a risk stratification tool to compute a CV risk factor based upon several risk stratification tables of reference. Additionally, the program includes a tool that incorporates ABPM analysis and allows the extraction of valuable information by monitoring blood pressure over a specific period of time. Finally, the reporting service summarizes the most relevant information in a set of reports that aid clinicians in their clinical decision-making process.

This article presents a protocol based on Hydra &#8212; a web-based system for clinical decision support that integrates a full and detailed set of functionalities and services required by physicians for complete cardiovascular analysis, risk assessment, early diagnosis, treatment, and monitoring over time.

Cardiovascular diseases (CVDs) are the leading cause of death throughout the world. The total risk of developing CVD is determined by the combined effect ...

International Expert Consensus and Recommendations for Neonatal Pneumothorax Ultrasound Diagnosis and Ultrasound-guided Thoracentesis Procedure

Pneumothorax (PTX) represents accumulation of the air in the pleural space. A large or tension pneumothorax can collapse the lung and cause hemodynamic compromise, a life-threatening disorder. Traditionally, neonatal pneumothorax diagnosis has been based on clinical images, auscultation, transillumination, and chest X-ray findings. This approach may potentially lead to a delay in both diagnosis and treatment. The use of lung US in diagnosis of PTX together with US-guided thoracentesis results in earlier and more precise management. The recommendations presented in this publication are aimed at improving the application of lung US in guiding neonatal PTX diagnosis and management.

Pneumothorax is a common emergency and critical disease in newborn infants that needs rapid, clear diagnosis and timely treatment. Diagnosis and treatment based on chest X-rays are associated with delayed management and radiation damage. Lung ultrasound (US) provides useful guidance for rapid, accurate diagnosis and the precise thoracentesis of pneumothorax.

Pneumothorax (PTX) represents accumulation of the air in the pleural space. A large or tension pneumothorax can collapse the lung and cause hemodynamic ...

Basophil Activation Test for Allergy Diagnosis

The basophil activation test (BAT) is a complementary in vitro diagnostic test that can be used in addition to clinical history, skin test (ST), and specific IgE (sIgE) determination in the evaluation of IgE-mediated allergic reactions to food, insect venom, drugs, as well as some forms of chronic urticaria. However, the role of this technique in the diagnostic algorithms is highly variable and not well determined.
BAT is based on the determination of basophil response to allergen/drug cross-linking IgE activation through the measurement of activation markers (such as CD63, CD203c) by flow cytometry. This test can be a useful and complementary tool to avoid controlled challenge tests&#160;to confirm allergy diagnosis, especially in subjects experiencing severe life-threatening reactions. In general, the performance of BAT should be considered if i) the allergen/drug produces false positive results in ST; ii) there is no allergen/drug source to use for ST or sIgE determination; iii) there is discordance between patient history and ST or sIgE determination; iv) symptoms suggest that ST may result in systemic response; v) before considering a CCT to confirm the culprit allergen/drug. The main limitations of the test are related to non-optimal sensitivity, especially in drug allergy, the need to perform the test no longer than 24 h after sample extraction, and the lack of standardization between laboratories in terms of procedures, concentrations, and cell markers.

The basophil activation test is a complementary in vitro diagnostic test for the evaluation of IgE-mediated allergic reactions based on the detection of basophil activation in the presence of a specific stimulus through the measure of activation markers by flow cytometry.

The basophil activation test (BAT) is a complementary in vitro diagnostic test that can be used in addition to clinical history, skin test (ST), and ...

In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is a devastating disease affecting preterm infants, characterized by intestinal inflammation and necrosis. Enteroids have recently emerged as a promising system to model gastrointestinal pathologies. However, currently utilized methods for enteroid manipulation either lack access to the apical surface of the epithelium (three-dimensional [3D]) or are time-consuming and resource-intensive (two-dimensional [2D] monolayers). These methods often require additional steps, such as microinjection, for the model to become physiologically translatable. Here, we describe a physiologically relevant and inexpensive protocol for studying NEC in vitro by reversing enteroid polarity, resulting in the apical surface facing outward (apical-out). An immunofluorescent staining protocol to examine enteroid barrier integrity and junctional protein expression following exposure to tumor necrosis factor-alpha (TNF-&#945;) or lipopolysaccharide (LPS) under normoxic or hypoxic conditions is also provided. The viability of 3D apical-out enteroids exposed to normoxic or hypoxic LPS or TNF-&#945; for 24 h is also evaluated. Enteroids exposed to either LPS or TNF-&#945;, in combination with hypoxia, exhibited disruption of epithelial architecture, a loss of adherens junction protein expression, and a reduction in cell viability. This protocol describes a new apical-out NEC-in-a-dish model which presents a physiologically relevant and cost-effective platform to identify potential epithelial targets for NEC therapies and study the preterm intestinal response to therapeutics.

In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis

This protocol describes an apical-out necrotizing enterocolitis (NEC)-in-a-dish model utilizing small intestinal enteroids with reversed polarity, allowing access to the apical surface. We provide an immunofluorescent staining protocol to detect NEC-related epithelial disruption and a method to determine the viability of apical-out enteroids subjected to the NEC-in-a-dish protocol.

Necrotizing enterocolitis (NEC) is a devastating disease affecting preterm infants, characterized by intestinal inflammation and necrosis. Enteroids have ...

Point-of-Care Ultrasound: A Review of Ultrasound Parameters for Predicting Difficult Airways

Airway management remains a crucial part of perioperative care. The conventional approach to assessing potentially difficult airways emphasizes the LEMON method, which looks for and evaluates the Mallampati classification, signs of obstruction, and neck mobility. Clinical findings help predict a higher likelihood of difficult tracheal intubation, but no clinical result reliably excludes difficult intubation. Ultrasound as an adjunct to clinical examination can provide the clinician with a dynamic anatomical airway assessment, which is impossible with clinical examination alone. In the hands of anesthesiologists, ultrasound is becoming more popular in the perioperative period. This method is particularly applicable for identifying proper endotracheal tube positioning in specific patient populations, such as those who are morbidly obese and patients with head and neck cancer or trauma. The focus is on identifying the normal anatomy, correctly positioning the endotracheal tube, and refining the parameters that predict difficult intubation. Several ultrasound measurements are clinical indicators of difficult direct laryngoscopy in the literature. A meta-analysis revealed that the distance from the skin to the epiglottis (DSE) is most associated with a difficult laryngoscopy. An ultrasound of the airway could be applied in routine practice as an adjunct to the clinical examination. A full stomach, rapid sequence intubation, gross visual anatomical abnormalities, and restricted neck flexibility prevent using ultrasound to assess the airway. The airway evaluation is performed with a linear array transducer of 12-4 MHz, with the patient in the supine position, with no pillow, and with the head and neck in a neutral position. The central axis of the neck is where the ultrasound parameters are measured. These image acquisitions guide the standard ultrasound examination of the airway.

A point-of-care ultrasound (POCUS) is a simple, non-invasive, and portable tool that enables dynamic airway assessment. Several studies have attempted to determine the role of ultrasound parameters as an adjunct to clinical examination in predicting difficult laryngoscopies.

Airway management remains a crucial part of perioperative care. The conventional approach to assessing potentially difficult airways emphasizes the LEMON ...

Multimodality Diagnosis of Mesenteric Ischemia

Early diagnosis of mesenteric ischemia remains challenging because mesenteric ischemia presents with no key symptoms or physical findings, and no laboratory data specifically indicates intestinal tissue ischemic status before necrosis develops. While computed tomography is the standard for diagnostic imaging, there are several limitations: (1) repeated assessments are associated with increased radiation exposure and risk of renal damage; (2) the computed tomography findings can be misleading because necrosis occasionally occurs despite opacified mesenteric arteries; and (3) computed tomography is not necessarily available within the golden time of salvaging the intestines for those patients in the operating room or at a place far from the hospital. This article describes a challenge to overcome such limitations using ultrasonography and near-infrared light, including clinical studies. The former is capable of providing not only morphologic and kinetic information of the intestines but also perfusion of the mesenteric vessels in real-time without transferring the patient or exposing them to radiation. Transesophageal echocardiography enables precise assessment of mesenteric perfusion in the OR, ER, or ICU. Representative findings of mesenteric ischemia in seven aortic dissection cases are presented. Near-infrared imaging with indocyanine green helps visualize the perfusion of vessels and intestinal tissues although this application requires laparotomy. Findings in two cases (aortic aneurysm) are shown. Near-infrared spectroscopy demonstrates oxygen debt in the intestinal tissue as digital data and can be a candidate for early detection of mesenteric ischemia without laparotomy. The accuracy of these assessments has been confirmed by intraoperative inspections and postoperative course (prognosis).

This article presents a multimodal approach that aims to overcome the limitations of traditional methods in detecting mesenteric ischemia and preventing bowel necrosis. The presented technique offers a promising solution by combining state-of-the-art ultrasonography with cutting-edge near-infrared light technologies.

Early diagnosis of mesenteric ischemia remains challenging because mesenteric ischemia presents with no key symptoms or physical findings, and no ...

Managing Gestational Vomiting with Ear Plaster Therapy

Ear Plaster Therapy as a Safe and Effective Treatment for Gestational Vomiting

Nausea and vomiting in pregnancy (NVP) are common symptoms that often complicate early pregnancy for many women. While clinical treatments such as fasting, fluid infusion, and nutritional support are conventionally applied to manage NVP, their effectiveness varies. However, traditional ear plaster therapy offers a promising alternative that effectively relieves symptoms and poses no known risk to the development of embryos or fetuses. This therapy is known for its ease of application, cost-effectiveness, and favorable outcomes. Previous studies have demonstrated the efficacy of combining ear plaster therapy with conventional treatments in alleviating symptoms of nausea and vomiting in pregnant women, surpassing the results achieved with conventional treatment alone. The protocol presented herein describes a method to relieve NVP using round, smooth, and hard cowherb seeds applied to specific ear points. These seeds are gently rubbed onto the surface of the ear, utilizing the principles of acupressure. By stimulating the designated ear points, this procedure aims to regulate the body's energy flow and restore balance, thereby reducing the severity and frequency of NVP. The application of cowherb seeds on specific ear points is a straightforward technique that healthcare professionals can easily implement or self-administered by pregnant women under appropriate guidance. Overall, ear plaster therapy presents a safe, effective, and economical approach for managing gestational vomiting, offering women a potential solution to alleviate their discomfort during pregnancy.

Author Spotlight: Alleviating Nausea and Vomiting in Pregnancy with Safe and Effective Auricular Acupuncture 

The presented protocol describes a traditional Chinese medicine treatment procedure known for its safety and effectiveness in managing gestational vomiting.

Nausea and vomiting in pregnancy (NVP) are common symptoms that often complicate early pregnancy for many women. While clinical treatments such as ...

Preparation for Ear Plaster Therapy to Treat Pregnancy Vomiting

Begin by preparing the ear plasters for ear plaster therapy. To do so, cut a medical adhesive tape into squares, each measuring 0.6 by 0.6 square centimeters. Then position one or two smooth cow herb seeds at the center of each tape square.Next, instruct the patient to assume a comfortable seated position. Examine the skin on the patient's ear to ensure that it is intact and free from any abnormalities before selecting the acupoints. To locate the potential acupoints, stabilize the ear oracle by pinching and lifting the ear helix backward and upward using the thumb and index finger of one hand, making sure the area is fully exposed.Then, using the sterile probe in the other hand, gently examine the patient's ear to identify sensitive pressure points near the six auricular points. The stomach or CO4, cardia or CO3, belly or AH8, shenmen or TF4, subcortex or AT4, and liver or CO12.

Ear Plaster Therapy for Nausea and Vomiting in Pregnancy

To begin, ensure the patient's ear auricle area has dried after disinfecting it with 75%alcohol, Then stabilize the auricle by pinching the ear helix with the thumb and index finger of one hand and lifting it backward and upward. Next, use the forceps to pick up the prepared ear plaster and place it onto the selected acupoint in the patient's ear, such that the cow herb seeds in the center of the plaster touches the selected point. Ask for the patient's assurance that the seed placement aligns with a sensitive or sore spot.Positioning the thumb and index finger in front of and behind the auricle. Apply moderate and tolerable pressure with rhythmic, pressing and relaxing motion to stimulate the cow herb seeds at the auricular site. Continue the rhythmic press and release at each site for three to five minutes, maintaining an even pace.Monitor the patient's overall condition for 15 minutes post-treatment. Instruct the patient or their family to stimulate the seeds by rubbing the concerned area three times a day, in the morning, at midday, and in the evening. After three days, remove the ear plaster and repeat the procedure on the opposite ear, alternating treatment between both ears.Compared to the control group, the experimental group, which received ear plaster therapy, had a significantly shorter average hospital stay and also required a significantly lower time for ketone bodies to turn negative. These suggested that ear plaster therapy can effectively reduce the treatment time for gestational vomiting. Prior to treatment, there were no statistically significant differences in PUQE scores between the experimental and control groups.However, after five days of treatment, both showed a significant reduction in the scores. But the score of the experimental group was significantly lower than that of the control group.