Alpaca Breeding Technologies | Transrectal Ultrasound in Alpacas

Exploration and Visualisation of Ovarian Structures in Alpacas by Transrectal Ultrasound

Jorge Reyna

MSc (Hons), MscVetSC (Sydney Univ.)

Lecturer in Higher Education - Learning Design

Faculty of Science

University of Technology Sydney

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INTRODUCTION

Transrectal ultrasonography has been used in a number of domestic species to observe the ovaries. This technique allows visualization of the ovarian structure in real time without direct manipulation (Pierson & Ginther, 1988) for the measurement of ovarian diameter, the number and size of follicles and corpora lutea , and the detection of pathologies such as cysts. Ultrasonography does not require anaesthesia, is non-invasive and repeatable (Clarke & Doughton, 1983). Initially it was developed for cattle (Pierson & Ginther, 1984; Reeves et al ., 1984) and is now the most popular method for the study of follicular dynamics in large domestic animals (Griffin & Ginther, 1992; Kahn, 1992).

Recently, high resolution probes (7.5, 9 and 10 MHz) have made possible a clearer visualization of the ovarian structures in domestic species. Now the technique is applied for the study of follicular dynamics in cattle (Pierson and Ginther, 1988; Knopf et al ., 1989; Bodensteiner et al ., 1995; Ginther et al ., 1997; Savio et al ., 1997; Sirois and Fortune, 1998), ewes (Noel et al ., 1993; Ravindra et al ., 1994; Leyva et al ., 1998; Evans et al ., 1999; Bartlewski et al ., 1999; Rubianes et al., 1999), goats (De Castro et al., 1999; Menchaca and Rubianes, 2002), camels (Tinson and McKinnon, 1992; Tibary and Anouassi, 2000) and in alpacas (Vaughan et al ., 2002; Reyna 2005).

Transrectal ultrasound in alpacas (TRUSA) is the most powerful tool when Multi Ovulation and Embryo Transfer programs (MOET) are applied. It allows the determination of previous ovarian status before starting a superovulatory treatment. Superovulation in alpacas in the presence of a dominant follicle may affect the yield and quality of embryos, as reported in other species like cattle and sheep. Another application of TRUSA is to determine the response of superovulatory protocols by counting the corpora lutea at the ovaries and to determine how many embryos we may expect to yield. TRUSA is also important before synchronisation of receptors. Finally, it is useful to determine reproductive pathologies like cystic ovarian disease that will affect the reproductive efficiency of the herd.

Much research conducted in female reproductive physiology in alpacas used TRUSA, but none of the studies present the technique step-by-step and documented with ultrasound images. This article describes for the first time in a simple manner how observations of the ovaries are made in alpacas, including imaging method, visualisation of structures and ultrasonographic patterns.

MATERIALS AND METHODS

Equipment

The ultrasound equipment used was a Sonovet 600 ultrasonic scanner (Medison Co., Ltd USA). This equipment has an automatic real-time linear array scanner operating in 4 different modes: B, BB, BM and M mode. The probe used was a linear-array dual transducer 5.0/ 7.5 MHz/ 40mm: Cable length 3 m (Medison Co., Ltd, USA).

Development of the technique

The training period to develop the ultrasound technique started in December 2005 and was completed in February 2006. Previous experience in transrectal ultrasound in cows and ewes allowed fast progress of the ultrasonographic sessions. For this purpose, a group of 12 females from 3-5 years old was used (Figure 1).

Procedure for scanning the ovaries

In the present report, two methods have been used to visualise ovarian structures. In both methods, animals were restrained in a holding crate in the sternal recumbency position. The first method introduces the probe using the hand (Figure 2) and the second method introduces the probe attached to a PVC pipe as reported in the ewe (Reyna, 2005). Both methods are effective for the observation of the ovaries

recipient alpacas

Fig 1 . Experimental animals at “La Hacienda” Marulan, NSW – Australia.

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Fig 2. Introduction of the probe with lubricated hand and localisation of the ovaries with fingers to lead the transducer to the ovarian surface in order to visualise the structures.


but require that the animals are acclimatised to the testing environment and accustomed to the technique.

Faeces were removed from the rectum with the right hand, and then 50 ml of a water-soluble, non-irritant viscous ultrasonic coupling medium (Ultrasonic Gel) was inserted into the rectum with a syringe to avoid damage to the mucosa (Figure 3). For the first method, upon introducing the hand with the transducer, fingers were used to palpate the ovaries and to lead the transducer onto the surface. For the second method, the ultrasound transducer, sheathed with a piece of PVC pipe (2 x 40 cm), was gently inserted into the rectum (20-35 cm). In both methods, the first visible structure located was the bladder, identified by its very dark contrast due to its liquid content. Passing the bladder it was possible to visualize the uterine body and horns. After location of the uterus, the transducer was rotated 90 degrees clockwise and 180 degrees counter clockwise across the reproductive tract until both ovaries were scanned. In some cases, upon application of the ultrasonic gel, the images were not clear and an extra cleaning of the rectum was performed by introducing 50-100 ml of water with a syringe to rinse out the contents. After scanning both ovaries, the probe was gently removed from the rectum.

Imaging method

The settings of the ultrasound equipment were kept constant where possible, but as the resolution of the images was not constant within and between animals, it was necessary to adjust the gain and contrast settings to obtain a

trusa

Fig. 3. Picture showing ultrasonic gel, syringe and transrectal probe (5.0/7.5 MHz)


clear image. The peristaltic movements of the digestive track also influenced the quality of the image obtained. After image freezing, some follicles lacked contrast and focus and sometimes had a solid appearance instead of the usual dark appearance. The best assessments were made during real time observations.

The ultrasound machine was connected to a handycam at all times and videos were recorded into DV tapes for further analysis. Videos were downloaded onto the computer to capture frames and digitise pictures on jpeg files using frame capture software (Power DVD). This methodology, although time-consuming, was the only way to confirm that the evaluations were accurate.

RESULTS

Exploration and visualisation of structures

Bladder and reproductive tract

After cleaning the rectum, and upon application of the ultrasonic gel, the transducer was gently inserted, usually between 20 and 35 cm deep in order to find the ovaries. The first visible structure found was the bladder, identified by its very dark contrast due to its liquid content (Figure 4). Passing the bladder it was possible to visualize the uterine body (Figure 5) and horns (Figure 6), which present a black pattern with a solid grey appearance around it corresponding to the muscular layer.

Ovaries

After location of the uterus the scanning surface of the probe was kept facing down the rectum. A rotation of approximately 90 degrees clockwise was then made and then one of 180 degrees counter clockwise to find the right and left ovary, respectively. The ultrasound image of the alpaca ovary was of a globular irregular shape with the surrounding tunica albuginea visible as a white pattern (Figure 7), with a clear and diffuse area in the inferior part that corresponded to the ovarian fimbria. The medullar layer of the ovary presented a hyperechoic diffuse pattern, the cortical layer was normoechoic or moderately hypoechoic and the hilus could be observed like a hyperechogenic line that penetrated the

alpaca bladder

Fig. 4. The bladder looks dark due to its liquid content and it is the first visible structure upon introducing the transducer onto the rectum.

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Fig 5. Traversal section of the uterine body in alpacas captured by transrectal ultrasound. The dark part corresponds to the lumen.

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Fig 6. Transversal section of the junction of the uterine horns

alpaca follicle

Fig 7. Alpaca ovary showing a globular irregular shape with a grey appearance with 5 and 3 mm follicles, respectively. The tunica albuginea surrounds the ovary as a white pattern.

ovary. On several occasions, when ovaries were not found at the first attempt, it was necessary to move the transducer forwards and backwards very gently inside the rectum until the structures became visible. In some cases, resolution was not good and it was necessary to rinse the rectum with water and apply more ultrasonic gel. The position of the ovaries varied slightly between scans according to the intestinal tract content. Every animal had individual characteristics of its ovaries. To make further evaluations easier it was necessary to become familiar with these characteristics during the scans performed.

Follicles

Upon location of the ovaries it was possible to visualise follicles by pressing the probe very gently against the ovarian surface. Follicles appeared as black circular structures surrounded by echogenic ovarian tissue, as fluid absorbs rather than reflects ultrasound waves (Figure 7). The follicular wall was normally very thin and well defined, especially in the area of contact with other follicles. Blood vessels appeared as black and circular structures, but were easy to identify by rotating the probe and observing their elongated appearance (Figure 8). In some cases, when the definition of the image was not clear, it was necessary to press the probe firmly against the ovary with very slow movements. The ultrasound machine resolved ovarian follicles with a diameter of 2 to 3 mm or greater, and larger antral follicles were easily tracked during serial scanning sessions. The measurements of the follicles were made once with the built-in callipers, making sure that the follicular wall

alpacas ultrasound

Fig 8. Blood vessels that look round and may be confused with follicles. When the probe is moving gently is possible to observe elongation.


was not included as this could be a source of error in finding the limit between the ovarian tissue and the follicular wall.

The ovulatory follicle was similar to any other large follicle and hard to identify if it was not tracked continuously before induction of ovulation in animals destined to be used as receptors for our ET program. There were some changes observed in the appearance of the ovulatory follicle, including an increase in the echogenicity of the antrum, and it was possible to notice a decrease in the resolution of the interior part closer to ovulation (20 hours after GnRH application).

Corpora lutea

After ovulation, the ruptured follicle becomes a corpus luteum (CL) which is responsible for the production of progesterone which maintains the pregnancy during the whole period of gestation in alpacas. The structure had a hyperechogenic shape, similar to the ovarian parenchyma but slightly less even. The corpus luteum (CL) appeared homogeneous with a normoechoic pattern and it was possible to determine its boundary with the ovarian tissue. The settings of the ultrasound machine were changed in order to visualize the CL, which required less contrast and brightness than follicles (Figure 9). The c orpus luteum in all the cases (n=5) presented a solid appearance and there were no cases of cavities as observed in the ewe (Reyna, 2005).

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Fig 9. Corpus luteum in a pregnant female at 11 months of gestation. The CL appears homogeneous and it is possible to determine its boundary with the ovarian tissue.


Placenta

During the development of the technique it was possible to visualise the placenta in some of the pregnant animals. After passing the bladder, the placenta can be visualised as a large dark structure due to its liquid content (Figure 10).

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Fig 10. Ultrasonographic image of the placenta showing a dark pattern.

CONCLUSION

TRUSA is a reliable technique to follow up follicular growth and atresia, to determine when ovulation occurs and the response to superovulatory treatments by observation of the corpora lutea . It can be used as a selection criterion in donors to increase the efficiency of the embryo transfer programs, as a diagnostic tool for reproductive pathologies, such as cystic ovarian disease, and also for pregnancy diagnosis. One of the main disadvantages is that the technique requires arduous training and an eye for detail. The development of the technique using a PVC pipe has an important application in the case of animals whose rectums are too tight to introduce the hand into, but this technique requires more skills and patience than manual technique.

ACKNOWLEDGMENTS

The author wishes to thank Luis and Suzy Bethencourt (La Hacienda – Marulan, NSW Australia) for providing the experimental animals and the ultrasound equipment, and also Peter Krockenberger for editorial assistance.

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Please cite as:

Reyna, J (2006). Exploration and Visualisation of Ovarian Structures in Alpacas by Transrectal Ultrasound. New Zealand Alpaca Association Magazine. Vol 3, Aug. 39-50 p.

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