ULTRASOUND IN EXTREME CONDITIONS – REPRODUCTIVE ASSESSMENTS OF WILD EURASIAN LYNX (LYNX LYNX) IN NORTHERN NORWAY
Painer J1, Arnemo JM2,3, Hildebrandt TB1, Jewgenow K1, Linnell JDC4, Odden J4, Göritz F1
1Leibniz Institute for Zoo and Wildlife Research (IZW), Alfred-Kowalke-Str. 17, 10315 Berlin,
2Hedmark University College, Campus Evenstad, Faculty of Applied Ecology and Agricultural Sciences, Department of Forestry and Wildlife Management, NO-2418 Elverum, NORWAY
3Swedish University of Agricultural Sciences, Faculty of Forest Sciences, Department of Wildlife, Fish and Environmental Studies, SE-901 83 Umeå, SWEDEN
4Terrestrial Ecology, Norwegian Institute for Nature Research, Tungasletta 2, 7485 Trondheim,
Reproductive ultrasound examinations were performed on immobilised free-ranging lynx (4 females, 3 males) in northern Norway, during the radio-marking season in February and March 2011. The portable ultrasound equipment was exposed to ambient temperatures as low as -25°C, without limitations of function in b-mode ultrasound and Doppler color flow technique. Results show no significant differences between these free-ranging lynx and captive Eurasian lynx (results from previously examined). The corpora lutea which physiologically persisted for almost a year, steadily produce elevated progesterone levels. This created the hypothesis of being the reason for turning a prolonged feline breeding season of several months, into a drastically shortened mating season in lynx. Examinations in February and March show that old corpora lutea had decreased their volume by half, indicating the start of luteolysis before and during next ovulation.
Ultrasound is a widely used tool in veterinary practice and gains more attention in routine and research diagnostics in zoo and wildlife medicine (Hildebrandt and Göritz, 1998). It has been shown to work well under clinical conditions and in the field under dry conditions and ambient temperatures (-5 to +40°C). However, there is no information on field application of use under harsh conditions down to -25°C. In February and March 2011, we joined the helicopter marking season of the Scandinavian lynx project, to get access to ultrasound data from free ranging animals. The project took place in northern Norway at 70° North latitude and 24° East longitude. Eurasian lynx (Lynx lynx) and wolverines (Gulo gulo) were immobilised with 4 mg medetomidine (Zalopine®, Orion Pharma Animal Health, Turku, Finnland) and 100 mg ketamin (Narketan 10®, Chassot, Dublin, Ireland) with a remote drug delivery system (Dan-Inject®) and reversed with 20 mg Atipamezol (Antisedan®, Orion Pharma Animal Health, Turku, Finnland) intramuscular (Arnemo et al., 2011). The lynx were captured for radio-collaring as part of an ongoing study of lynx ecology (e.g. Linnell et al., 2010).
We were especially interested in documenting the ovarian status of female lynx immediately prior to the onset of the mating season in comparison to data collected with the same examination protocols in captive lynx (unpublished data). Furthermore we wanted to test the ultrasound equipment for field application. It has been shown that lynx have some peculiarities that are unique among felids concerning their ovarian activity (Göritz et al., 2009). Lynx have physiologically persisting corpora lutea all year round, which prevents a normal ovarian cyclicity and ensures a very strong seasonality.
Material and methods
Four free-ranging adult female lynx, three adult male lynx and two female (one subadult, one pregnant) wolverines were examined. A portable Ultrasound machine (Voluson i, General Electronics Company Healthcare) was used, equipped with a 12 MHz transcutaneous linear probe, real-time 2D ultrasound and Doppler color-flow sonography were applied. Size of ovaries and testicles, number and size of corpora lutea (Cl), size of adrenal and anal glands were measured. Furthermore, the structure and texture of kidneys were evaluated.
Adaptation of materials to harsh conditions
Self-sustainable heating pads were used to prevent freezing and cracking of the liquid crystals from the ultrasound monitor (LCD) and the piezo-crystal elements inside the probe. The equipment was exposed to ambient temperatures down to -25°C, at even lower temperatures (down to -36°C) examinations were conducted indoors. A non-transparent and waterproof blanket was used to provide shade for the screen and allowed the ultrasound image to be viewed in the very bright surrounding of the snow covered tundra and protect the electronics from falling and drifting snow. The machine itself was transported in a medium sized Peli™ case, insulated with 3 cm polyethylene-plates and humidity-absorbant patches inside.
Due to cold conditions with temperatures down to -36°C, the fur was not shaved for examinations – which would of course be optimal for improving ultrasound-skin contact and wave penetration. Instead, the hair was parted to get a small “window” for improved skin contact and a few drops of 70 % alcohol was used for receiving a direct contact without producing too much body-heat loss, due to evaporation. After ultrasound examination the fur was dried off with paper towels.
Results and discussion
The capture period of the Scandinavian lynx project in the north of Norway, enabled us to obtain ultrasound data from free-ranging lynx. Results from these free-ranging lynx were compared with captive lynx, which were sampled throughout the year in Germany (unpublished data). The samples collected in Norway were taken during a very important time period: the pre-ovulatory time. Results show no significant differences between captive and free-ranging lynx in their ovarian structure, size and amount of corpora lutea or follicles (captive: 3.5 Cl’s per ovary, free-ranging: 3.0 Cl’s per ovary). In addition, in one wolverine pregnancy could be diagnosed by visualisation of two foetuses. Cardiac and umbilical blood flow, placental size, placental attachment and organ development were evaluated for the first time.
Female Eurasian lynx show persistent corpora lutea throughout the year (Göritz et al., 2009) with elevated progesterone levels (Jewgenow et al., 2010), which is as far as we know unique amongst felids. Ultrasonography done in northern Norway in February and March showed a decreased size in old corpora lutea during the pre-ovulatory period. The inner part of the corpora lutea showed transformation and a collapse of structure, seen as dense heap of structural elements on the ultrasound image, indicating the change into a corpus albicans (figure 1). From mid-March on, large follicles (on average: 2 follicles per ovary; 0.74 cm x 0.48 cm, figure 2) and significantly bigger and denser corpora lutea were found on the ovaries, reflecting pre-ovulatory and post-ovulatory period.
Fig. 1: Ovary of an Eurasian lynx (Lynx lynx) in February. Arrow indicates dense structure in one of the smaller, older corpora lutea, showing the structural collapse of a corpus luteum. Second image shows the structures with help-lines.
Fig. 2: Ovary of an Eurasian lynx around the period of ovulation in March. Follicles of different sizes (black) and a Corpus luteum (white line) can be seen. The second picture shows the structures with helplines.
This was the first time there has been a chance to perform ultrasound in freshly ovulated lynx ovaries. Old corpora lutea were on average 0.49 x 0.49 cm, fresh corpora lutea were on average 0.89 x 0.89 cm in size. The fresh Cl’s were not only bigger in size, they also showed increased vascularisation measured by color flow ultrasound (figure 3). This indicates regression (luteolysis) in the old Cl’s and increased metabolite activity in the fresh Cl’s. These data prove, that new ovulation can occur while some old Cl’s are still present (figure 2).
Further research on ovarian topography using 3D-ultrasound is in progress, to document the complete annual ovarian cycle for lynx. To improve the knowledge on assisted reproduction techniques on lynx genus, further follow up will be done this year to strengthen our hypothesis:
- persisting Cl’s cause a very narrow breeding season
- being induced or facultative spontaneous ovulators and
- the strategy of natural and medical induced luteolysis in lynx.
Fig. 3: Ovary of Eurasian lynx, Vascularisation visualised with Doppler colour flow. Fresh and dense in structure: corpus luteum seen in the middle (big ellipse). Small and old copora lutea around (small ellipses). Second images shows the structures with help-lines.
We would like to thank the BMBF (German federal ministry of education and research) for funding (No.: BMBF 033L046). Furthermore we thank the Scandinavian Lynx project (www.scandlynx.no) for the possibility of joining this year’s capture event. Scandlynx’s research activity is mainly funded by the Research Council of Norway and the Norwegian Directorate for Nature Management.
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