The authors have declared that no competing interests exist.
Conceived and designed the experiments: JP KJ FG. Performed the experiments: JP FG KJ MD TBH JMA. Analyzed the data: JP. Contributed reagents/materials/analysis tools: JMA MD FG TBH KJ JDCL JO. Wrote the paper: JP KJ FG JDCL JMA. Endocrine laboratory analysis: MD KJ. Fieldwork and project logistics in Scandinavia: JMA JDCL JO JP.
Felids generally follow a poly-estrous reproductive strategy. Eurasian lynx (
The Eurasian lynx (
Many felid species are known to express a poly-estrous reproductive pattern: being able to mate several times a year
The ultrasound approach produces a high quality image for non-invasive soft tissue examinations and can be used to obtain clear information about the status of reproductive organs
The present study used detailed longitudinal data of healthy lynx females held in zoos. The study includes the evaluation of the formation of CLs after ovulation in pregnant and non-pregnant animals, the luteal function during and after pregnancy or pseudo-pregnancy, as well as the luteal regression before next ovulation. To exclude that the results were an artifact of working with captive animals under artificial conditions, we took advantage of access to free-ranging lynx to conduct control examinations.
The examinations of captive lynx were performed when the animals were immobilized for other reasons, including veterinary monitoring, minor health intervention or due to captive animal management reasons. The methods applied, and the study-design, were in agreement with the animal ethics and welfare committee at the Leibniz Institute for Zoo and Wildlife Research (IZW, Berlin, Germany. No: 2010-01-01). The study of free-ranging lynx was conducted within the frames of the Scandinavian Lynx Project, Scandlynx (
This study was conducted on ten captive female lynx examined 2-6 times (three animals were examined twice, one animal three times, two animals four times, three animals five times and one animal six times) each between April 2010 and July 2012. The reproductive history of each individual is listed in
Ten free-ranging lynx were examined in February and March in northern Norway in 2011 and 2012
The captive lynx were darted inside their enclosures using a blowpipe and a 3 mL dart syringe equipped with a 22 gauge dart-needle (1.2×38 mm) (all from DanWild LLC, Dan-inject dart guns, TX, USA). An initial dose of 0.06 mg/kg Medetomidine (0.1%, Domitor, Orion Corporation, Espoo, Finland) plus 4.0 mg/kg Ketamine (Ketamine 10%; Essex GmbH, Munich, Germany) was used. During anesthesia, the animals were supplied with intranasal medical oxygen and intravenous isotonic NaCl-Infusion (0.9%, Braun, Tuttlingen, Germany). Respiration, heart rate, pulse-oxymetry (Nellcor, CA, USA), rectal temperature and eyelid-reflexes were constantly monitored.
The free-ranging lynx were darted from a helicopter and immobilized with medetomidine plus ketamine, using previously established protocols from Arnemo et al.
Blood was withdrawn from the free-ranging and captive lynx from the
In the captive lynx, small areas of fur were clipped in an area cranio-lateral to the second mammary complex (counted from caudal). Ethanol (70%) and ultrasound gel (Aquasonic 100, Parker Lab, NJ, USA) were poured on to achieve skin-contact for the ultrasound probe. An ultrasound laptop (Voluson i, GE-Health, Austria, Zipf) equipped with a 12 MHz linear probe (12 L – RS) and a 6–16 MHz volume probe (RSP 6 – 16 RS) were used for imaging. By applying brightness (B-) mode, the ovaries were visualized and length, width and breadth of the ovaries as well as maximum diameter of each CL were measured on screen. Volume was calculated using the mathematical formula “
Due to the harsh climate conditions (cold temperatures) and time constraints, only 2D ultrasound and Doppler modes were applied to the free-ranging lynx. The animals were not clipped; instead the fur was combed to reveal a small skin window.
Estrogens (E2), P4 and the prostaglandin F2α metabolite (PGFM) were analyzed in serum samples using in-house enzyme immunoassays
All tests were conducted using the software package R.2.14.1
The different reproductive stages in female lynx were classified as pro-estrus, estrus, met-estrus, pregnancy, pseudo-pregnancy, lactation and prolonged di-estrus (
The ultrasound images (in b-mode) of lynx ovaries demonstrate different stages of ovarian activity. The white scale-bar indicates 1 cm. Asterisk indicates follicles, empty arrows show new CLs; full arrows show old
Schematic diagram of the development of follicles and CLs within a reproductive year in pregnant and non-pregnant lynx. P4, E2, PGFM in ng/mL are shown with lines.
cycle | CL per | CL tissue | ovarian | diameter. | number | ||||
stage | N | E2 | P4 | PGFM | ovary | (cm2) | volume | A |
follicles |
10 | 0.59±0.34 | 2.65±2.77 | 2.59±0.81 | 3.26±1.69 | 0.88±0.64 | 2.18±1.44 | 0.22±0.04 | 1.53±2.43 | |
4 | 1.49±0.04 | 2.08±0.70 | 1.23±0.45 | 2.50±2.17 | 0.78±0.62 | 1.55±1.05 | 0.24±0.00 | 1.83±1.72 | |
3 | 0.78±0.86 | 13.07±8.1 | 1.46±0.44 | 2.17±2.32 | 1.02±1.61 | 1.93±1.19 | 0.23±0.08 | 1.00±1.26 | |
26 | 0.32±0.21 | 4.68±3.45 | 1.82±0.91 | 2.66±1.43 | 0.65±0.48 | 1.74±1.03 | 0.20±0.05 | 0.37±0.76 | |
3 | 0.56±0.14 | 84.05 | 2.61±1.18 | 4.17±1.17 | 1.55±0.66 | 3.80±2.33 | 0.29±0.05 | 0.67±1.21 | |
±83.85 | |||||||||
2 | 0.18–0.44 | 3.14– | 4.41–1.48 | 2.75±0.50 | 1.66±0.92 | 2.35±0.96 | 0.25±0.03 | 0.00±0.00 | |
170.38 |
Mean values ± S.D. of various ovarian and serum parameters in free-ranging (N = 10) and captive (N = 38) lynx examinations during the various stages of the reproductive cycle, based on a 2 year study (2010–2012). E2 = serum estrogens in ng/mL, P4 = serum progesterone in ng/mL, PGFM = serum prostaglandin F2alpha in ng/mlL, CL =
One captive female was a juvenile (19 month); accordingly we observed small ovaries (0.54 cm3 and 0.10 cm3) without follicular or luteal activity (
Pro-estrus was found in lynx examined just before the mating season in early spring. These females had already undergone at least one reproductive cycle in a previous year (Ncaptive = 5, Nfree-ranging = 5). Several hypo-echoic CLs were detectable on the ovaries using ultrasound (
In one captive female a second behavioral estrus was observed by the keepers about 2 months after her normal seasonal estrus. This female was kept without a male throughout. During ultrasound examination one rather small follicle was seen in each of her ovaries (0.27 and 0.34 cm diameter,
Estrus occurred at the end of February until mid-March for the captive lynx (N = 2) and at the end of March for the free-ranging lynx (N = 2). Behavioral signs of estrus (calling, rolling) lasted between 5 and 10 days for the captive animals observed in this study. Nevertheless, the zoo-keepers occasionally observed calling from the males or females already 3–4 weeks in advance. One captive female was in her first estrus. In this case we observed one follicle on each ovary (0.96 and 0.78 cm,
Met-estrus, thus the period shortly after ovulation, was detected in three animals with clearly depicted ovulation scars (
After the post-estrus luteal formation period, freshly formed fully functional CLs were judged to be a clear indication of a recent ovulation. The fresh CLs differed in their sono-morphology from the old CLs of previous cycles (
In pregnant females the secretion of P4 increased rapidly after ovulation. The CL tissues appeared hyper-echoic compared to pseudo-pregnant new CLs at the same time (
After weaning (day 100 postpartum) the CLs appeared similar to CLs of non-pregnant females (Ncaptive = 24, Nfree-ranging = 2) at the same time (
Values of P4 during prolonged di-estrus were significantly correlated with the intensity of vascular support measured using the diameter of the
Prolonged di-estrus in lynx outside breeding season in December. Empty triangle points at CLs from this years' ovarian cycle. Full triangle points out one old CL, at least two years old.
One captive animal was examined after its reproductive senescence (at 19 and 20 years of age). Steroid hormones measured were at very low levels with E2 = 0.39 ng/mL and non-detectable P4. Both ovaries appeared similar to juvenile ovaries, non-active and without functional bodies and only a minor vascularization.
Repeated examinations were performed in six captive animals encompassing all non-pregnant cycle stages (pro-estrus, estrus, met-estrus, prolonged di-estrus). No significant differences emerged when comparing the concentrations of serum P4, E2, PGFM, ovarian volume, the intensity of vascular support (diameter A.
By repeated ultrasound and hormonal examination in captive lynx, our study confirmed the physiological persistence of CLs derived from ovulations (
The lynx reproductive cycle shows new and so far unknown dynamics in luteogenesis and luteal regression amongst felid species (
In the past, all felids were believed to be exclusively coitus induced ovulators. However, later studies have shown that some cat species are able to ovulate spontaneously. Most felids express a combination of induced and spontaneous ovulation with individual variability
Henriksen et al.
During the lifespan of an animal there is a limited period for fecundity. This period usually starts at puberty (in female lynx mostly in their second year
The hormonal support mechanism of persistent CLs still needs to be identified. In the domestic cat, prolactin is known to be a luteotrophic factor
The evolution of a mono-estrous reproductive system in lynx could have primary (the ancestors of felids were mono-estrous) or secondary (adaptive) origins. A phylogenetic constraint, however, seems unlikely as mono-estrous breeding is a very unusual pattern among felids in general, and the most primitive member of the genus
The lynx reproductive cycle seems to be a rather non-plastic system. We found no difference in reproductive pattern between either wild and captive lynx or between central and northern European populations. The only anticipated difference was found concerning the timing of breeding seasons, which is most likely latitude, and therefore delayed photoperiod, dependent. However, the captive and free-ranging populations both showed the same reproductive strategy i.e. being mono-estrous due to physiologically persistent, constantly P4 secreting CLs, without plasticity regarding this phenomenon.
The main conservation implications of these results is the confirmation that lynx are indeed mono-estrous. The fact that ovulation appears spontaneously implies that pregnancy is very dependent on having access to a male during the crucial period of estrus. Anything that reduces access to males in this narrow window would result in an entire year's reproduction being lost. Many of the larger lynx populations are subject to hunter harvest in some form, and hunting is normally conducted in the late winter because of the hunter's dependence on good snow conditions
A second conservation implication concerns the ability of lynx to adapt to environmental changes. The physiology described in this paper indicates that lynx will have relatively little ability to adjust birth dates as it appears to be a remarkably non-plastic system. However, the full implications of this are not clear, because we do not yet know the environmental cues for the timing of ovulation in lynx.
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We thank M. Rohleder and K. Paschmionka for performing all hormone analysis and K. Carnaby, Dr. J. Saragusty, and Dr. A. Courtiol for their very helpful support. Many thanks to the participating zoos for their motivation to contribute to the lynx research (TP Stendal, TP Essehof, TP Bischofswerda, TP Thale, TP Johannismühle, Zoo Stralsund, Zoo Magdeburg). We furthermore thank P. Segerström, T. Strømseth, and E. Segerström for catching and collaring the lynx. Many additional people have contributed in the field.