Feminizing Syndrome in African Lions: Behavioral, Phenotypic and Hormonal Aspects
Feminizing syndrome can be caused by a number of reasons such as neoplasias in testes, adrenal glands, or in the pituitary, which lead to abnormal levels of sexual steroids, luteinizing hormone or GnRH or due to alterations in the sensitivity of the target organs to the hormones. This syndrome is not common with zoo and wild animals. However, in certain circumstances, it can compromise the management of a group or even breeding programs in zoos and in the wild. It is very important to investigate the possible causes of this syndrome in order to make correct decisions with respect to the individuals and species involved. This syndrome may interfere with normal behavior causing disruption of the relationships inside animal groups. The present paper reports a clinical case of feminizing syndrome in an adult male African lion (Panthera leo) from the São Paulo Zoo Foundation in São Paulo, Brazil.
A 9-year-old male African lion, with a successful breeding record, kept in the São Paulo Zoo began to present a marked reduction of the mane and became totally maneless in a few weeks. The mane is a classical secondary sexual characteristic in male lions. This male used to fight with other males of the group to gain access to the cycling females and after the observed phenotypic change, it was noticed that the lion was no longer fighting. Instead, other males were observed fighting against each other for an opportunity to mount the maneless feminized male, which was seen accepting those mountings several times. Another interesting behavior observed was that the feminized male lion began to help the females of the group take care of the cubs.
The feminized male lion was restrained in a squeeze cage and blood samples were collected twice per day (morning and afternoon) for 3 days to measure serum testosterone and cortisol levels. This procedure was repeated with six normal male lions for comparison. The feminized male was then anesthetized with a combination of ketamine hydrochloride (Vetasept®, Fort Dodge Animal Health, São Paulo, Brazil; 10 mg/kg IM) and xylazine hydrochloride (Rompun®, Bayer, São Paulo, Brazil; 0.1 mg/kg IM). As soon as the lion was immobilized, a blood sample was collected and immediately afterwards was injected with the GnRH agonist lecirelin (Gestran Plus®, Arsa SRL, Buenos Aires, Argentina; 25 µg IM). Blood samples were collected every hour after the lecirelin injection until the lion’s recovery. The same protocol was repeated with a phenotypically and behaviorally normal male lion. During sedation, we conducted an andrologic examination involving the inspection of external genitalia, checking the presence of spicules in the glans, as an indirect parameter of normal testosterone level, and palpations of the testes for consistency evaluation. The testis volume (V) was estimated by measuring the length (L) and width (W) of each testis using sliding calipers and then calculating the volume using the formula V=L×W2×0.524.3 The two values for each male were added together to provide a volume for the combined testes. The andrologic examination was repeated with six normal male lions. Four months after this procedure, the feminized male was again restrained with the same anesthetic protocol and at this time we injected LH from purified porcine pituitary extract (Lutropin-V®, VetPharm, ON, Canada; 25 mg IM) right after sampling blood, and serial samples of blood were collected every hour until the recovery of the animal. The same procedure was also performed with the normal male lion. Testosterone and cortisol levels for all of the blood samples were measured with a commercially available radioimmune assay (Coat a Count®, Diagnostic Products Corp., Los Angeles, CA, USA).
The feminized male presented a total testicular volume of 25.50 cm3, smaller than the mean value obtained from the six normal males in São Paulo Zoo 50.96±9 cm3 (p<0.01). It was also smaller than the data from literature, 69.8±14.9 cm3 in the lions from Ngorongoro Crater and 87.8±6.4 cm3 in the population from Serengeti.1 The testes were harder in consistency than in the normal males. There were very tiny spicules in the glans of the feminized lion when compared with the clearly evident ones in the normal males. The average testosterone level for the feminized lion was 0.10 ng/ml. This value was far below the mean level obtained from the six normal males, 0.72±0.65 ng/ml (Student’s t-test, p<0.001). The values for the six normal lions are comparable with data found in literature 1.85±0.53 ng/ml for adult captive lions in Indian zoos2 and 1.13±0.25 ng/ml and 0.96±0.36 ng/ml obtained from free-living lions in Serengeti plains and in the Ngorongoro Crater, respectively.1 After the injections of GnRH agonist, it was seen that the testosterone level of the normal male began to rise in the first hour reaching a peak of 1.15 ng/ml at 5 hours. The injection of LH in the normal male only showed a sharp rise after 5 hours, reaching a peak of 0.54 ng/ml at 8 hours. The feminized lion showed no significant change in testosterone level in either of the challenges, never reaching more than 0.33 ng/ml after the GnRH and then 0.14 ng/ml after the LH. No significant differences in the cortisol levels were observed between the feminized lion, 12.30±2.87 µg/dl and the normal male, 13.41±2.92 µg/dl.
The results showed a clear difference between the testosterone levels of the feminized lion and the normal males. The possible source of this anomaly was investigated by the injection of the two different hormones in order to check whether the testes or the pituitary gland were involved. After the stimulation with GnRH agonist and LH, only the normal male had an increased in the testosterone production, while the feminized male showed no response. With the first hormonal challenge with GnRH agonist, we intended to induce an endogenous release of LH with an expected rise in testosterone levels, which could mean that there was a possible lack of LH production by the pituitary in the abnormal lion. In the second treatment with a supposedly high dose of exogenous LH, we were checking the level of sensitivity of the Leydig cells to a direct stimulation by LH. In both cases, the absence of a response in the feminized male and the clear response of the normal male strongly suggested that there is a lack of testicular sensitivity to LH stimulation in the abnormal lion. We expected that the time elapsed between the hormone injections and the increase of testosterone in the normal lion would be shorter with the LH treatment than with the GnRH agonist. This is because we were skipping one of the phases in the classic hypothalamic-pituitary-gonads hormonal axis; however, we observed the opposite. One possible explanation for these results could be that with the GnRH treatment the responses were due to the release of endogenous LH. However, with the injection of a heterologous LH (porcine) we observed a delay in the increase of testosterone, which could be due to a lower sensitivity of the specific receptors in the Leydig cells. Another possibility is that the dosage of LH was too low. There are no proven procedures for this kind of hormonal challenge in male lions, and there is no information in the literature, thus, the experiment was simply empirical. The lack of an increase in testosterone levels in the feminized male lion could be explained by a theoretical breakdown in the LH receptors in the Leydig cells, which is difficult to confirm. The cortisol levels showed that there was no significant difference in the stress levels between both animals during the procedures.
This work characterized a clinical case of feminizing syndrome by means of phenotypical, behavioral, and hormonal evaluations and suggested the etiology based in two hormonal challenges, although it was impossible for us to confirm the lack of testicular LH receptors. Further investigations on a molecular basis are determinant to confirm this theory.
The authors would like to thank to the staff of São Paulo Zoological Park Foundation for their generous assistance. We are also in debt with the Technopec Cons. Com. e Rep. Ltda, São Paulo, Brazil for providing the GnRH agonist and Regina Célia Rodrigues da Paz for the donation of the LH.
1. Brown, J.L., M. Bush, C. Packer, A.E. Pusey, S.L. Monfort, S.J. O’Brien, D.L. Janssen, D.E. Wildt. 1991. Developmental changes in pituitary-gonadal function in free-ranging lions (Panthera leo leo) of the Serengeti Plains and Ngorongoro Crater. Journal of Reproduction and Fertility. 91:29–40.
2. Shivaji, S., D. Jayaprakash, and S.B. Patil. 1998. Assessment of inbreeding depression in big cats: testosterone levels and semen analysis. Current Science. 75:923–929.
3. Wildt, D.E., M. Bush, S. O’Brien. 1993. New Opportunities in Animal Health Sciences Training Manual. NOAHS Center, Washington, DC.