ULTRASONOGRAPHY OF THE EXTERNAL REPRODUCTIVE ORGANS IN TOM

This study aims to delineate the tissue structure of male cat external reproductive organs using ultrasound with different types of consoles and transducers frequencies through indirect method. External organ of 5 tom weighing 2-3 kg was evaluated using ultrasound with stationary console (linear transducer, 10 MHz) and portable console (linear transducer, 6.5 MHz), transcutaneously. The results showed that both console and transducer could not visualize the structure of organs sized less than 10 mm through direct method. However, the indirect method using stationary console with a 10 MHz transducer enable to visualize the organ and depicts clearly the internal organ structure such as penis and its parts, scrotum and its constituent layers, caput and cauda of epididymis, and testicles. Furthermore, portable console with 6.5 MHz transducer was still able to provide an adequate image of those organs. In general, fibrous tissues such as tunica vaginalis, tunica Dartos, funiculus spermaticus, and urethra were visible in ultrasonogram as hyperechoic structures, while soft tissues such as the parts of testicles and penis were visible as hypoechoic structures. Tissue containing fluid such as urethral lumen was visible as anechoic structure. In conclusion, indirect ultrasound imaging method was sufficient to visualize the organs sized less than 10 mm using 3-15 MHz transducer in which was currently widely used in animal health care clinics. ____________________________________________________________________________________________________________________


INTRODUCTION
Ultrasonography could visualize structure the architectural details of soft tissues and organs.The image produced in sonogram is created using highfrequency sound wave and is displayed as real-time slices of images similar to histological images (Pavlin and Foster, 1998).High-frequency sound wave of 20 MHz is commonly used in the medical services for dermatological examination (Gniadecka and Quistorff, 1996).This method known as dermatosonography (Jemec et al., 2000) while 30-100 MHz was used for eye examination (Pavlin and Foster, 1998).However, such high-frequency transducer is not available in most medical facilities.The most commonly available transducer in medical facilities are 3-15 MHz transducers (Mantis, 2008) that used for obstetrical examination and imaging of other internal organs.
The sound wave produced and captured by transducers consists of several parts namely Fresnel zone, focal zone and Fraunhofer zone (Noviana et al., 2012;Mattoon and Nyland, 2014).Target organ examination is performed at focal zone to obtain an image with adequate resolution.However, the limitation of 3-15 MHz transducers is the location of Fresnel zone more than 10 mm, hence the target organ could not be depicted clearly.Ultrasonography examination of scrotum in humans (Hebert et al., 2012) and large animals (Constantinescu, 2005) with a scrotal size of > 30 mm is easy because most of the organ is located at the focal zone (Mantis, 2008).However, difficulty arises when examining small animals and pets that owing small sized of organs (Dyce et al., 2009).Although ultrasonography has been introduced since 1978 (O' Grady et al., 1978), reports on normal and abnormal image of small mammals with tissue width < 20 mm are rare (Redrobe, 2001).The health status of small mammals in zoos or their habitat is very important for its survival (Carey and Johnson, 1995), hence there is a need for new approaches on the application of ultrasonography imaging.
Internal organs sized < 10 mm such as ovaries (Davidson and Baker, 2009a), testicles and penis (Davidson and Baker, 2009b) and their parts are difficult to be visualized through ultrasonography.Reproductive organs examination for pets and wild animals inside conservation areas are important to monitor their health status and to diagnose certain abnormalities.Although there has been reports of ultrasonography imaging on the reproductive organs of male dogs (Davidson and Baker, 2009b) and thin tail sheep (Ulum et al., 2013), there has been no detailed report on external reproductive organs of toms.New ultrasonography method is needed to visualize tissues and organs sized < 10 mm using the currently available diagnostic equipment.This study reports the result of ultrasonography on external reproductive organs of tom which included scrotum, testicles, penis and prepuce, using consoles equipped with transducers that having difference frequencies through indirect imaging method.This method could be used as a model for ultrasonography of small mammals with tissue width of < 20 mm.The result of this study would improve diagnostic imaging not limited on pets and small mammals but also for conserved animals such as reptiles, amphibians, fishes and other small exotic animals.

Research Animal
The animals used in this study were 5 clinically healthy tom aged 1-2 years old weighing 2-3 kg with scrotal circumference was 6.0-7.5 cm and scrotal diameter ±10 mm.Uncooperative tom was anesthetized to aid the imaging process.As much as 0.025% atropine sulfate at dose of 0.02-0.04mg/kg body weight was injected subcutaneously 10-15 minutes before anesthetized (Mann et al., 2011).The tom was then anesthetized using 10% ketamine at 10 mg/kg body weight and 2% xylazine at 2 mg/kg body weight intramuscularly (Fossum, 2013).After anesthesia, the tom was laid on their left comfortably on the examination bed.The tom that did not need anesthesia was handled and restrained physically with firmly but comfortably during imaging at their preferred position (laid on its back, recumbent or prone).

Ultrasonography Imaging
Ultrasonography imaging of external reproductive organ of tom was performed using a stationary ultrasonography console unit (Mirror 2, Landwind Medical, China) with 10 MHz linear transducer and a portable ultrasonography console (SonoVet, Meditech Equipment, China) with 6.5 MHz linear transrectal transducer (Table 1).The hairs on the scrotum, prepuce and perineum were not shaved (Figure 1).Ultrasonography gel was applied on the genital area and was flattened so that the hairs, skins, and the spaces between hairs are completely covered by the gel.Transducer was directly pressed on the scrotum transcutaneously from dorsal to ventral through the caudal area.The views used on testicles and penis were oblique, longitudinal, and transverse; then the skin and prepuce were evaluated.This direct ultrasonography method is commonly used nowadays so that the image obtained in the focus area could be seen clearly.Direct imaging method was performed on testicles and epididymis while indirect imaging method was performed on penis, prepuce, and scrotum.Indirect method was performed by placing the imaging target on focus area so that target organ is not directly in contact with the transducer.The obtained image was documented as a video and the next documented image was then analyzed to be further interpreted.The image was then interpreted based on the structures appearance such as black (anechoic), gray (hypoechoic), and white (hyperechoic).

Data Analysis
The ultrasonographic image of external reproductive organs and its parts was analyzed descriptively.

Reproductive Organs of Tom
The reproductive organs of tom was consist of 4 parts (Constantinescu et al., 2007;Davidson and Baker, 2009b;Dyce et al., 2009).The first part is primary reproductive organs such as testicles, epididymis, vas deferens, spermatic cord and tunica.The second part is accessory glands such as prostatic glands and bulbourethral (Cowper) gland.The third part is penis as the copulatory organ, and lastly urethra which is also part of the urinary system.External reproductive organs are located outside of the body and it is visible by bare eyes.External reproductive organs in toms include: 1) scrotum, which contains testicles and epididymis, and 2) penis which is enveloped by prepuce (Davidson and Baker, 2009b;Dyce et al., 2009).External reproductive organs of tom are located at the perineum, under the tail and rectum.Penile body is linked to ischiocavernous muscle and penile retractor muscle (Dyce et al., 2009).Scrotal sac emerges from the perineum with penis at its ventral side (Figure 2).

Technique of Ultrasonography Imaging
Ultrasonography imaging works by utilizing of high-frequency sound wave, which is divided into 3 zones namely Fresnel zone, focal zone, and Fraunhofer zone (Matton and Nyland, 2014;Noviana et al., 2012).Fresnel zone is where complex diffractions patterns occur, located nearest to the transducer as the sound source.Focal zone is where the sound wave is most focused, producing an image with the best resolution.Fraunhofer zone is the part where sound wave diverges and the resolution of the image gradually decreases (Figure 3a).Ideally, the target organ of ultrasonography is directly positioned in the focus zone by placing the transducer in the imaging area, especially for large organ or dense tissues.Focal zone position can be set to be close to the sound source in the transducer.However, visualizing small organ with direct technique would be difficult because of its small size.Organs sized < 10 mm will be automatically visualized in Fresnel zone, hence the image produced will not have any diagnostic value.Ultrasonography on small organs < 10 mm was done using indirect approach in this study (Figure 3b) to position the target organ in the focus zone.Indirect ultrasonography imaging technique on penis, for example, was done by placing the transducer at dorsal surface of the scrotum to visualize penis which is located at its ventral side (Figure 1a, Figure 1d, and Figure 1f).
Ultrasonography imaging of cat external reproductive organs (testicle, epididymis, scrotum, prepuce, and penis) in this study were done using indirect technique by utilizing console unit and transducer with different frequencies, 10 MHz and 6.5 MHz.The internal structure of organ tissues can be visualized by using ultrasonography imaging.Image produced shows different results as sonogram with difference of echogenicity, especially in detailed basic tissue structure such as testicle, epididymis, scrotum, prepuce, and penis.

Sonogram of Prepuce
Figure 4 shows the sonogram of prepuce (p) which appears hypoechoic compared to the more hyperechoic scrotum (s).Prepuce lumen (lp) appeared as anechoic in contrast to the more hypoechoic prepuce (p), glans penis (gp), and body of penis (bp).Sonogram at 10 MHz transducer could delineate and distinguish prepuce more clearly compared to 6.5 MHz transducer.Scrotal skin (k) located near the transducer could not be imaged clearly while the components of the parts inside the focus area could be delineated clearly.
Prepuce is a skin that acts as cover of glans penis (Dyce et al., 2009).The thickness of prepuce is < 2 mm, hence indirect imaging is the technique of choice.In ultrasonography, prepuce appeared hypoechoic compared to the hyperechoic scrotum because prepuce is only composed of tubular sheets of skin.This skin consists of internal and external lamina.Prepuce lumen appeared as an anechoic structure between the hypoechoic prepuce skin tissues, glans penis, and penile body because skin tends to repel sound waves (Figure 4 and Figure 5).Sonogram with higher frequency transducer can differentiate the structures that made up prepuce tissues clearly.

Sonogram of Penis
Figure 5 shows sonogram of penis that consisted of glans penis (gp), body of penis (bp), and urethra (u).Glans penis (gp), and body of penis (bp) appeared hypoechoic.Urethra (u) appeared hyperechoic while its lumen appeared anechoic.The components of penis could be seen more clearly in the image from sonogram with 10 MHz transducer compared to 6.5 MHz transducer.Scrotal skin (k) located near the transducer could not be imaged clearly while the components of the parts inside the focus area could be delineated clearly.
Penis is a copulatory organ that located at the ventral of scrotum and consists of body of penis, glans penis, and urethra (Dyce et al., 2009).Toms have coneshaped glans penis; the tip points caudally and has penile spikes (Johnston et al., 2001).The spike numbers about 120-150 pieces (Davidson and Baker, 2009) and act as vaginal simulator or ovulation inductor for queen during copulation (Dyce et al., 2009).For a very small penis or organ (< 5 mm), indirect ultrasonography is the technique of choice.Glans penis and body of penis appear hypoechoic because of its horn epithelium covered connective tissues that reflect of sound wave (Figure 4 and Figure 5).Short os (bone) of penis at the tip of glans penis appears distinctly on sonogram.Urethra appears hyperechoic with anechoic urethra lumen inside because it is filled with fluids.Sonogram equipped with higher frequency transducer can clearly differentiate all basic structure of an organ.

Sonogram of Epididymis
Figure 6 shows sonogram of epididymis as more hypoechoic structure compared to testicle (t).Caput epididymis (cpe) appeared more hyperechoic compared to cauda epididymis (cde).The boundary between epididymis and testicular body (t) could be seen more clearly in the image from 10 MHz transducer compared to the image from 6.5 MHz transducer.Scrotal's skin (k) located near the transducer could not be imaged clearly while the components of the parts inside the focus area could be delineated clearly.
Epididymis is elongated ducts that located at the medial part of testicles and it has a function as a spermatozoa maturation site (Dyce et al., 2009;Hebert et al., 2012).Epididymis can be identified around testicles as a homogenous structure and appears hypoechoic compared to the testicles (Mantis, 2008).Ultrasonography of epididymis can be performed using either direct or indirect technique.In ultrasonography, caput epididymis appears as an anechoic structure.The size of duct is smaller in caput epididymis compared to cauda epididymis (Figure 6).In lower frequencies (6.5 MHz), it appears as a combination of hypoechoic and anechoic structure.At caput epididymis, anechoic appearance is more dominant compared to cauda epididymis.Scrotal skin appeared as hypoechoic to hyperechoic line.

Sonogram of Testicle
Figure 7 shows sonogram of testicle, the main organ that produces spermatozoa and the main reproductive organ in male animals.At the dorsolateral of testicle (t) there is cauda epididymis which is connected to spermatic duct.Testicles appeared anechoic at 10 MHz transducer while they appeared hypoechoic at 6.5 MHz transducer.The mediastinum (ms) in the middle of testicle (t) appeared hyperechoic.The boundary between scrotum (s) and testicular body (t) could be seen more clearly using 10 MHz transducer compared to 6.5 MHz transducer.Scrotal skin (k) located near the transducer could not be imaged clearly while the components of the parts inside the focus area could be delineated clearly.

Figure 1 .
Figure 1.Technique of ultrasonography imaging of the external reproductive organ of tom.a= Transversal view at the base of dorsal scrotum, b= Transversal view at apex of caudal scrotum, c= Transversal view at the base of ventral scrotum, d= Longitudinal view at the base of dorsal scrotum, e= Longitudinal view at the base of ventral scrotum, f= Oblique view at the base of dorsal scrotum

Figure 2 .
Figure 2. External reproductive organ of tom after given ultrasound gel.a= Side view, b= Back view

Figure 3 .
Figure 3. Classification zone of ultrasonography imaging.a= Transducers with different frequencies, b= Different imaging method

Table 1 .
Specification of brightness mode (B-mode) ultrasonography console unit