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Contents

  1. Top of page
  2. Contents
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgment
  9. Conflicts of interest
  10. References

Assisted reproduction technologies are essential for propagating endangered wild felids. Artificial insemination (AI) has been reported in several wild feline species, but pregnancy rates are low, partially owing to failures of current hormonal stimulation protocols. Therefore, this study describes the application of reliable methods to monitor ovarian activity and the development of an effective hormonal protocol to induce oestrus and ovulation in African lions. Application of porcine FSH and porcine LH was shown to be effective for inducing follicular growth and ovulation, and this regimen appeared to be superior to protocols described earlier in terms of ovulation and fertilization rates. Furthermore, non-surgical AI was performed successfully in lions, and uterine-stage embryos were collected and cryopreserved. African lions may serve as a valuable model to develop assisted reproduction for propagation of relic zoo populations in the critically endangered Asian lion or Barbary lion.


Introduction

  1. Top of page
  2. Contents
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgment
  9. Conflicts of interest
  10. References

African lions (Panthera leo) normally breed well in captivity, necessitating the application of contraception methods as a management tool in some zoos (Munson, 2006). However, the wild population is highly fragmented and suffers progressively from geographic isolation and inbreeding (O'Brien et al. 1987; Bertola et al. 2011). Within Africa, the West African population is particularly endangered. Epidemic diseases as described earlier in Serengeti lions (Roelke-Parker et al. 1996) can also contribute to the extinction of existing wild populations. The Asiatic lion (Panthera leo persica) is actually critically endangered in the wild (Singh et al. 2002) and breeding success in zoos is very limited. Assisted reproductive technologies may be essential for propagating endangered wild felids in captivity (Wildt and Roth 1997). Successful artificial insemination (AI) has been reported in only 10 wild feline species to date using surgical and non-surgical techniques. Pregnancies were achieved by surgical intrauterine insemination (laparoscopy or laparotomy) in eight species (tiger, snow leopard, cheetah, clouded leopard, puma, ocelot and tigrina (Moore et al., 1981; Barone et al. 1994; Howard et al. 1996; Swanson et al. 1996; Howard and Wildt 2009), by transvaginal insemination in the Siberian tiger, lion and leopard (Bowen et al. 1982; Dresser et al., 1982; Chargas e Silva et al. 2000) and by ultrasound guided transcervical insemination in cheetah and Amur leopard (Goeritz, unpublished data). Before AI, oestrus and ovulation have to be induced. Although there are a variety of studies on ovarian control for assisted reproduction in domestic and wild cats (Pelican et al. 2006), there has been minimal research performed in protocols to induce oestrus and ovulation for AI in lions.

Developing successful assisted reproductive techniques requires knowledge of the female reproductive cycle and precise control of ovarian activity. Therefore, the aim of the present study was to develop reliable methods to monitor ovarian activity and an effective hormonal protocol to induce oestrus and ovulation to perform AI in African lions.

Material and Methods

  1. Top of page
  2. Contents
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgment
  9. Conflicts of interest
  10. References

Animals

Four 6-year-old nulliparous female lions (Panthera leo), born and housed at Givskud Zoo, Denmark, were used for this study. These lionesses were not exhibited to the public and were kept indoors, individually housed and separated from males. All animals were healthy and sexual cycle behavioural activity was documented previously.

The dominant breeding male (proven breeder) was chosen as the semen donor for the AIs; this male was housed on exhibit with a group of three to six females.

Anaesthesia

To perform the subcutaneous application and removal of a hormonal implant (see 'Hormonal treatment') as well as for AI and embryo retrieval, all animals had to be anaesthetized four times. General anaesthesia was achieved by injection of 60 μg/kg medetomidine (Zalopine; Orion Corporation, Espoo, Finland) and 3 mg/kg ketamine (Ketamine 10%; Essex GmbH, Munich, Germany) via dart gun. The duration of anaesthesia ranged from 30 to 40 min. For longer procedures (e.g. semen collection by rectal electro-ejaculation), the animals were intubated and anaesthesia was maintained with 2–3 Vol% isoflurane with an oxygen flow rate of 4–7 l/min (Penlon, Abingdon, UK). Antipamezole (Antisedan; Orion Corporation; 5 mg/1 mg medetomidine administered) was given to antagonize the effect of medetomidine.

Ultrasonographical and endocrinological monitoring of ovarian activity

Transabdominal ultrasonography was performed (Voluson i, GE equipped with a 4–8 MHz volume transducer and 12 MHz linear transducer) to visualize the female reproductive tract. Size of the ovaries, number and size of follicles and/or corpora lutea (CL's) were measured. Blood was collected and estradiol (E2) and progesterone (P4) were measured in blood serum as described before (Goeritz et al. 2009).

Hormonal treatment

All animals received an etonogestrel implant (68 mg; Implanon NXT, Organon, the Netherlands) subcutaneously to down regulate ovarian activity and synchronize the animals. Sixty-four days later, the animals were immobilized again and the implants were removed. The ovarian status was assessed ultrasonographically and endocrinologically during each anaesthetic event. Two different hormonal protocols were initiated (day 0). Two animals (group I) received a single dosage of 750 IU eCG followed by 100 IU hCG 4 days later. The other two animals (group II) received pFSH on four consecutive days (50/100/100/50 IU) followed by injections of 50 IU pLH on two consecutive days. All hormones were applied intramuscularly via dart without sedation. The treatment protocols, timelines and procedures performed are summarized in Table 1.

Table 1. Treatment protocols and hormones applied to control ovarian activity and to induce follicular growth as well as ovulation for artificial insemination in lions
DayGroup I (n = 2)Group II (n = 2)
  1. a

    One etonogestrel implant (68 mg; Implanon NXT, Organon, the Netherlands) per animal applied subcutaneously;

  2. b

    eCG (Pregmagon, IDT, Dessau-Rosslau,Germany) and hCG (Ovogest, Intervet, Germany);

  3. c

    Application via dart using a blow pipe.

  4. d

    pFSH and pLH (Sioux Biochemicals, Sioux Center, Iowa, USA).

−64Immobilization, ultrasonography, blood sample, Implantsa inserted
0

Immobilization, ultrasonography, blood sample, Implantsa removed

750 IU eCGb i.m.c

Immobilization, ultrasonography, blood sample, Implantsa removed

50 IU pFSHd i.m.c

1100 IU pFSHd i.m.c
2100 IU pFSHd i.m.c
3 50 IU pFSHd i.m.c
4100 IU hCGb i.m.c 50 IU pLHd i.m.c
5 50 IU pLHd i.m.c
6Immobilization, ultrasonography, blood sample, semen collection & AI
13Immobilization, ultrasonography, blood sample & embryo collection

Semen collection, artificial insemination and embryo collection

On day 6, all animals were anesthetized and ovarian status was evaluated again. Non-surgical AI was performed with fresh semen obtained by electroejaculation (Seager Model 14, Dalzell, Marshal, USA) from a breeding male housed nearby. Semen obtained was diluted with semen extender (1 : 1, TEST medium without glycerol) immediately and evaluated microscopically (Goeritz et al. 2006). To inseminate all females, the total volume was divided into four equal aliquots. The equipment and technique used for non-surgical AI was the same as described recently for uterine lavage in large cats (Hildebrandt et al. 2006). Special catheters (Gynétics Medical Products N.V., Lommel, Belgium, #6000 lion, Ref. Nr.: 1355F) were introduced transvaginally into the cervix under sonographical guidance (transrectal sonography using a portable ultrasound system; EUB 405, Hitachi GmbH, Germany equipped with a 7.5 MHz fingertip probe; F345). Seven days later (day 13), the final evaluation of ovarian activity and embryo collection was performed surgically in three animals (n = 2 group I, n = 1 group II). The oviducts and uteri were flushed retrograde and the flushing medium (TCM 199 containing Earle′s salts, Waurich et al. 2010) retrieved was scanned for embryos microscopically.

Results

  1. Top of page
  2. Contents
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgment
  9. Conflicts of interest
  10. References

With ultrasonography, ovaries were identified in dorsal recumbency caudal to the kidneys. Follicles and CLs could be clearly distinguished. Etonogestrel implants appeared to cause down regulation and synchronization of ovarian activity in all four animals (Fig. 1). Ovaries were small, and no CLs and only few small follicles (diameter < 1 mm) could be visualized by ultrasound at the time of implant removal. The serum concentrations of E2 and P4 were also low with 7.34 (±1.56) pg/ml and 0.53 (±0.33) ng/ml, respectively.

image

Figure 1. Ultrasonographic image showing an down-regulated ovary (black circle) 64 days after subcutaneous application of an implant containing 68 mg etonogestrel (Implanon NXT, Organon, the Netherlands). No corpora lutea or large follicles are visible; however, the ovarian cortex is distinctly hypoechogenic in contrast to the medulla, associated with minimal follicular activity (small antral follicles with a diameter of ≤1 mm, black arrows). Scale bar = 5 mm

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Both hormonal protocols induced follicular development (Fig. 2), endometrial activity and clear signs of oestrus in the lions. Mean numbers of follicles per animal detected sonographically at the time of AI were 11.5 (group I) and 14.0 (group II). Concentration of E2 measured in blood serum was 28.76 (±6.19) and 54.38 (±36.24) pg/ml in group I and group II, respectively. No CLs could be detected sonographically, which was confirmed by low P4 concentrations (group I: 0.46 ± 0.01 ng/ml; group II: 0.87 ± 0.58 ng/ml) in blood serum at the same time.

image

Figure 2. Ultrasonographic image showing an active ovary containing multiple Graafian follicles (black arrows) at the time of artificial insemination after hormonal treatment using pFSH and pLH. Appearance of mature follicles as well as elevated E2 concentration in blood serum indicates successful induction of follicular growth. Scale bar = 5 mm

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Ovarian ultrasonography during embryo collection revealed the appearance of CLs in both groups (Fig. 3). Mean numbers of CLs detected were 6.5 and 10.0 per animal in group I and group II, respectively. Hence, ovulation rate ranged from 56.5% (group I) to 83% (group II) with corresponding mean P4 values of 11.91 ng/ml (group I) and 29.15 ng/ml (group II).

image

Figure 3. 3D sonogram of an ovary during embryo collection showing multiple corpora lutea (CLs) as spherical structures (numbers) clearly distinguished from surrounding ovarian parenchyma (appears more light coloured). Scale bar = 5 mm

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Five fractions of semen with a total volume of 2.4 ml, an overall motility of 73% and progressive motility of 68% were collected by electroejaculation. Each female received one ml of extended semen containing 72 × 106 sperm cells. Whereas only two unfertilized oocytes and one two-cell stage embryo were flushed out of the uterus in group I, three apparent embryos at the late morula stage were recovered from one of the animals from group II (Fig. 4). One morula was fixated and evaluated under phase contrast; the other two embryos were cryopreserved using a vitrification protocol developed for cat oocytes (Mikołajewska et al. 2012).

image

Figure 4. Microscopic images showing an unfertilized oocyte, a two-cell stage embryo and a morula stage embryo, recovered from the uterus of hormonally stimulated lions 7 days after artificial insemination

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Discussion

  1. Top of page
  2. Contents
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgment
  9. Conflicts of interest
  10. References

Low pregnancy rates following AI in most endangered cats may be partially attributed to failures of current hormonal stimulation protocols. Species differences may be significant and can easily lead to ovarian hyper-stimulation (Pelican et al. 2006). Hormonal protocols using eCG/hCG have proven capable of inducing follicular development and ovulation in a variety of wild felids (Chargas e Silva et al. 2000; Pelican et al. 2006). This study confirms that these hormones are also effective in lions. The second protocol applying porcine FSH and porcine LH was shown to be successful for inducing ovarian activity and ovulation in lions for the first time. Based on our limited sample size, this protocol seems equal to or possibly superior to eCG/hCG in terms of ovarian stimulation and ovulation rate. Our findings are in accordance with studies showing that feline FSH/LH is most analogous to porcine FSH/LH (Crichton et al. 2003). However, the necessity of daily injection of pFSH for four consecutive days and pLH for another two consecutive days was impractical and stressful for the lions. Future studies should focus on implementation of feline FSH/LH regimens and less invasive administration modes for wild felids, such as mini-pump implants or lipidic long-acting formulations. Pre-treatment with etonogestrel, a progesterone analog, appeared to help synchronize the cycles prior to gonadotropin treatment. The absence of CLs after the removal of the implants seemed to improve ovarian response to the gonadotropins (Pelican et al. 2006).

On day 7 after AI, unfertilized oocytes and embryos could be retrieved from the uterus, although recovery success was low relative to the number of CL observed via ultrasonography. Severe side effects such as ovarian cysts, ovarian hyperstimulation, hydrosalpinx or mycometra (Hildebrandt et al. 2006) could not be observed. The embryos collected in this study were cryopreserved by vitrification. The post-thaw survival and the developmental capacity to more advanced stages have not been evaluated yet, although blastocyst formation is considered a more accurate measurement of oocyte quality than cleavage rate (Pelican et al. 2006). For final assessment of the hormonal protocol, further in vivo studies are needed to determine whether this protocol is suitable to produce live offspring.

High-resolution transabdominal ultrasonography was very useful for monitoring ovarian and uterine activity. In comparison with normal 2D-greyscale ultrasonography, 3D ultrasonography was superior to evaluate ovarian topography and exact number of follicles and CLs (Painer et al. 2012). However, guidance of the insemination catheter into the cervix and monitoring of uterine deposition of the semen were possible with transrectal ultrasonography (Goeritz et al. 1997) only. The vagina and cervix are located in the bony pelvis and therefore not detectable by transabdominal ultrasound. In terms of practicability and risk of impaired wound healing or secondary infection, this non-surgical insemination technique is more adaptable for field application than the laparoscopic AI technique described in other wild felids (Howard et al. 1996).

Conclusions

  1. Top of page
  2. Contents
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgment
  9. Conflicts of interest
  10. References

In this study, non-surgical AI was used to inseminate four African lions, allowing the recovery of embryos for cryopreservation. Application of pFSH and pLH after down regulation of ovarian activity with etonogestrel represents one option to induce follicular development, oestrus and ovulation in this species. Further studies are needed to determine whether this protocol can result in production of live offspring. The African lion may serve as a valuable model for developing assisted reproduction methods for conservation of relic zoo populations in the critically endangered Asian lion or Barbary lion (Burger and Hemmer 2006).

Acknowledgment

  1. Top of page
  2. Contents
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgment
  9. Conflicts of interest
  10. References

The professional animal care of the lions was provided by the staff of the Givskud Zoo, Denmark. Collected embryos were evaluated and further handled by Romy Hribal, Caterina Wiedemann and Natalia Mikolaewska, all from IZW Berlin. They also provided the microscopic pictures of the embryos. We thank Nga Nguyen, Jette Ziep, Marlies Rohleder and Katrin Paschmionka for their technical assistance. The study was partly funded by BMBF 033L046.

Conflicts of interest

  1. Top of page
  2. Contents
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgment
  9. Conflicts of interest
  10. References

The authors do not have any conflicts of interest and they do not have affiliations that may be perceived as having biased the presentation.

References

  1. Top of page
  2. Contents
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgment
  9. Conflicts of interest
  10. References
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