Follicular Stasis in Captive Chelonians, Testudo spp.

Stuart McArthur, BVetMed, MRCVS

Holly House Veterinary Surgery, 468 Street Lane, Moortown, Leeds LS17 6HA, UK

Abstract: This paper reviews nonnal female chelonian reproductive endocrinology. Clinicopathological data, treatment regimes, and the outcome of 10 presumed cases of follicular stasis are described in captive chelonians, Testudo spp. presented over a period of 3 years. Endocrinopathy associated with follicular stasis and some potential causes offollicular stasis are explored.

Key Words: Chelonian, anorexia, endoscopy, ultrasonography, follicular stasis, proligestone, ovariectomy.

INTRODUCTION

Follicular stasis is where vitellogenesis fails to progress through to ovulation and oviposition, and consequently affects the well-being of the animal (Backues and Ramsay 1994). Follicular stasis appears to be relatively common in captive oviparous lizards (Divers 1996), but is not yet reported in captive chelonians. (Table 1.)

Endocrinology ofnormal reproduction in the female tortoise

Progesterone

e In reptiles, a distinct corpus luteum is fonned following ovulation, and available evidence suggests that it secretes progesterone (Bentley 1998; Kuchling 1999b).

e There is evidence that progesterone is one ofthe major steroids synthesized by the chelonian ovary (Klicka and Mahmoud 1977), and there is evidence that the corpus luteum of the chelonian is capable ofsynthesizing progesterone (Klicka and Mahmoud 1972; Klicka and Mahmoud 1973; Chan and Callard 1974; Callard and others 1976).

• Progesterone was shown to completely inhibit ovulation and reduce pituitary size, oviduct size, and follicle size in the turtle, Chrysemys pieta (Klicka and Mahmoud 1977).

$ Negative feedback inhibition of the release of a pituitary derived trophic honnone is suggested by Bentley (1998). Bentley further proposes that estrogens and progesterone can influence the hypothalamic release of reptilian gonadotropins in a manner similar to mammals.

e A potential physiological role of progesterone in the regulation of clutch size ~nd maintenance ofgravidity has been proposed (Klicka and Mahmoud 1977). In Seeloporous eyanogenys, Callard and others (1972) propose that progesterone prevents follicular development byboth direct action on the hypothalamus and possibly peripherally, resulting in inhibition ofvitellogenesis.

zOO 1 I8roceeaings • ~ssoGiation of FReptilian ana ~mpnibian FReptiles 75 • Rostal and others (1998) found that progesterone levels in the Galapagos tortoise, Geochelone nigra, displayed a sharp surge during the mating period that coincided with ovulation. e However, Licht (1984) suggests that various chelonians, including the green sea turtle, Chelonia mydas, do not maintain postovulatory levels of progesterone as expected and therefore the role ofprogesterone in chelonians may vary between species. Estrogen

(3 In reptiles, estrogen stimulates vitellogenesis, the production ofIipophosphoproteins by the liver, and their incorporation into the egg (Callard, and others 1972; Licht 1979; Duvall and others 1982; Kuchling 1999b).

• Ovarianmaturation and follicular growthinthe Galapagos tortoise coincides with elevations in estradiol levels (Rostal and others 1998).

• Ovarian maturation and follicular growth in Dermochelys coriacea follows an initial elevation in estradiol levels (Rostal and others 1996).

Testosterone e In Geochelone nigra, testosterone levels were elevated during the matingperiod immediately prior to ovulation. This rise was presumed to relate to receptivity of female (Rostal and others 1998). A similar rise in serum testosterone is observed in D. coriacea (Rostal and others 1996) and Lepidochelys kempii (Rostal and others 1997). However, boththese species demonstrate unique elements ofnesting behavior [such as arribada and yolkless eggs], and these in tum may relate to unique reproductive endocrinology.

Gon~dotropins

• The development of the ovary, its secretion of steroid honnones such as estrogen and testosterone, and ovulation, appear to be controlled by follicle stimulating honnone (FSH) or a nonspecific gonadotrophin. Pregnant mare serum gonadotrophin (PMSG), which has primarily FSH activity, was shown by Klicka and Mahmoud (1977) to promote chelonian ovarian growth.

• Mammalian FSH has been shown to induce ovulation in several species of lizards and Bentley (1998) assumes that an endogenous gonadotrophin has this effect in most reptiles. Licht and Papkoff(1974) specifically demonstrated this in Chelydra serpentina.

• Bentley (1998) reports that pituitary FSH and lutenizing honnone (LH) have both been identified in reptiles and negative feedback by estrogen on the pituitary gland release of gonadotropins is proposed by Bentley (1998).

,. :l ... ,• -., .. ." ,:l ,e .. -., .. Potentialfactors influencing chelonian vitellogenesis and ovulation

• Light intensity and photoperiod - day length and its rate of change (Bartholomew 1959; Peaker 1969)

e Ambient temperature and its rate ofchange (Peaker 1969; Licht 1972; Licht 1984; Duval and others 1982; Bentley 1998) • The influence ofpheromones on female chelonians, such as those released from male mental glands (Rose 1969; Winokur and Legler 1975)

• The act ofmating (Kuchling and Razandrimamilafiniarivo 1999)

• Further factors such as rainfall, moisture, humidity, nutrition and food supply (Licht 1984; Duval and others 1982)

• Potentially influenced by endogenous factors such as the balance of progesterone, testosterone and estrogen and/or thyroid hormones; as in avian species (Millam 1977)

Is follicular stasis in chelonians a genuine pathological condition?

To date a diagnosis ofchelonian follicular stasis remains presumptive and it is therefore a diagnosis of exclusion. This underlines the importance of a full clinical examination and a thorough assessment ofthe anamnesis. As follicular stasis is a chronic disease, all efforts should be made to rule out other concurrent disease.

It is unclear if follicular stasis is merely a secondary sign of other health problems, or a specific condition in its own right. There is an apparent correlation between prolonged folliculogenesis, hepatic lipidosis, chronic anorexia, and nonspecific illness. The author believes that follicular stasis is a condition in its own right. It relates to an endogenous, physiological need for partial or complete induction ofovulation through mental gland or other pheromones and contact (mating, butting or biting) from a male tortoise. Therefore, a failure of induced ovulation by restricted exposure to suitable male tortoises results in follicular stasis and disease.

Prolonged duration of reproductive arrest while at an advanced stage of follicular development, visualization ofa large number ofadvanced follicles within each ovary, and prolonged anorexia, all suggest follicular stasis. However, there is limited reproductive data available to define what is "normal" with respect to ovarian cycles in captive female chelonians. This is especially true of Testudo spp. It is unclear that it is valid to extrapolate hormonal and other reproductive findings from different chelonian speciese Ultimately, until there is a larger database of "normal" reproductive physiological data for each species encountered, the diagnosis/condition ofstasis must remain presumptive. This paper hopes to reveal data that might help to validate follicular stasis as a plausible diagnosis in future, especially with respect to captive Testudo spp.

DIAGNOSIS

History/Anamnesis

All aspects ofhusbandry and nutrition should be investigated. Cases presented to this author have invariably been isolated, mature, female tortoises, often they have beenprovided with low standards of nutrition and husbandry. Follicular stasis appears to be a chronic disease. Signs might arise months after onset of the disease; consequently this disease does not appear to show strong seasonality. Cases have been presented in summer, autumn, winter, and spring.

•••• I • II! • II! II! • I •• Clinical signs

Lethargy, anorexia, abdominal distension, and weight gain have been reported in lizards with follicular stasis (Backues and Ramsay 1994; Divers 1996). Cases eventually diagnosed as in follicular stasis, are generally presented with prolonged anorexia and absence offeces. The keeper commonly reports total anorexia and an absence offeces for periods of3-7mo. Clinical pathology

Differentiationbetweenperiods ofnonnal follicular development and follicular stasis is notpossible using a single blood sample. Trends over time must be represented and an adequate database of nonnal infonnation in captive Testudo spp. is not yet available.

Hypercalcemia (elevations intotal calcium), hyperalbuminemia, and elevations intotal proteinoccur in females undergoing vitellogenesis. It is possible that ionized calcium levels are simultaneously subnonnal.

til Hypercalcemia occurred in 8/10 cases • Hyperalbuminemia occurred in 7/9 cases

til Elevated total protein occurred in 7/9 cases • Alkaline phosphatase was elevated in 8/8 cases

til Moderate anemia occurred in 7/7 cases • Leukopenia occurred in 5/7 cases e Heteropenia occurred in 5/7 cases

Cases that have become chronically anorexic, are in concurrent liver failure, or have concurrent metabolic bone disease may not show all of these changes. These findings presumably relate to hyperestrogenism and/orstarvation. Ifthe follicular stasis is uncomplicated, otherhematological and biochemical parameters may be within normal ranges.

Wilkinson (2000) suggests the disease may also be characterized by acidic urine (Innis 1997) and raised blood and urine ~-hydroxybutyrate [Ketosis] (Christopher and others 1994),as these changes are consistent with catabolism in herbivores.

Cholesterol and triglyceride levels may become elevated during vitellogenesis (Derikson 1979). Normal levels are poorly defined. However, similar elevations might also occur during hepatic lipidosis, which appears to be intimately associated with follicular stasis, and numerous other conditions causing anorexia in chelonians.

Estrogen, testosterone and other honnone assays may prove to be offuture use. Ultrasonography

Data on the ultrasonography assessment ofnonnal annual follicular cycles ofUK captive female Testudo spp. is limited. Several breeding and nonbreeding females have beenmonitored using serial ultrasonography. During late autumn and spring, moderate numbers offollicles (generally less than 20), 15-22mm in diameter, are considered to be nonnal in mature female Testudo spp. Where the numbers ofmedium sized follicles (5-15mm), and large follicles (15-22mm), become in excess of

I • ., .., ...... 50, follicular stasis or ovulatory failure should be suspected. If sequential ultrasonography in Testudo spp., over several weeks, demonstrates >20 mature ovarian follicles, 15-22mm diameter, follicular stasis is suggested. In cases offollicular stasis, some follicles may not seem to progress to ovulation, but might regress and become resorbed.

Ultrasonography supported the diagnosis in 4/5 cases diagnosed. One animal did not have adequate anatomical access to the prefemoral acoustic window to allow scanning. Endoscopy

Endoscopy examination of mature females in follicular stasis may reveal ovaries with 20-50 follicles, 15-22mm diameter. It is not easy to differentiate between normal folliculogenesis and stasis from a single examination. An accurate count of developed coelomic follicles is often impractical.

Endoscopy supported the diagnosis in 8/10 cases. Exploratory coeliotomy

Exploratory coeliotomy in cases offollicularstasis will reveal a coelomiccavitypacked with 50-200 follicles, 15-22mmdiameter. It is hard to differentiatebetweennormal ovarian activity and follicular stasis without further supportive history and evidence that follicular progression was absent.

TREATMENT AND PREVENTION

Follicular stasis has been reported to regress inlizards (Backus and Ramsey 1994), butit is unknown ifspontaneous resolution might also occur in chelonians. Surgical and medical treatments utilized all appear to have both advantages and disadvantages. These include improvements in environment, nutrition, and social interactions whereverpossible, and specificmedical orsurgical treatment aimed at resolving inappropriate ovarian activity.

Improvements in environment

Photoperiod andtemperature management shouldbeassessed and formalized. All chelonians should be given a suitable annual photo period cycle (Jones 1978).

Improvements in nutrition

Potential nutritional disorders should be identified and managed. Hepatic lipidosis is a consistent finding in cases of reproductive stasis. Cases should be managed as though concurrent hepatic lipidosis is present. Social interactions

Tortoise ovulation may be induced by the presence ofa suitable male tortoise. Discourage keepers from maintaining mature tortoises as isolated females. It is unknown if a meeting with a male tortoise, or a mating, will stabilize female reproduction. Females that are mixed with appropriate males every 2-3yr may maintain normal reproductive function better than those that are not.

200 1 Proceedings • '£xssociation of Reptilian and kXrnpnibian Reptiles 79 ~edicaltreatnBent

Rosskopfand Woerpel (1983) used medoxyprogesterone acetate at a dose of22mg/kg body weight to suppress ovarian activity in the Californian desert tortoise, Geochelone agassizii, and 20mg/kg by Bennett (1998). Frye (1991) suggests that twice yearly dosing with a mammalian dose of medoxyprogesterone acetate will completely suppress reptilian follicular activity.

Proligestone (Delvosterone; Intervet) has been given at 20mg/kg. It successfully reduced follicular activity in a Testudo hermanni diagnosed as having follicular stasis.

No adverse effects are described in the literature as a result ofmedical suppression of chelonian follicular activity using progestogen, but adverse effects seem plausible. Cases suspected to have follicular stasis are also likely to have hepatic lipidosis. The return ofa large amount offollicular material to a compromised liver, following progestogen therapy and successful follicular resorption, may be detrimental to health. Potential interactions between insulin and progesterone might result in diabetes. In some cases offollicular stasis in Testudo spp., repeated progesterone therapy has not stimulated follicular regression. Subsequent ovarioectomy was required.

Thyroid hormone appears necessary for normal ovarian regression in birds. Supplementation in thryoidectomized starlings induced photorefractoriness and induced gonadal regression (Millam 1997). Therefore it is plausible that thyroid function may also influence efficacy ofproligestone therapy in chelonians. The relationship between testosterone levels and ovulation is also poorly understood, and it is similarlyplausible that circulating levels ofotherhonnones such as testosterone might influence proligestone efficacy.

A conclusive outcome regarding the efficacy of proligestone has not yet been determined, but proligestone appears better able to suppress ovarian activity in early cases or injuveniles reaching puberty, and it may therefore prove a suitable drug to prevent the condition, especially in isolated juvenile females.

Surgical treatment

Follicle aspiration was suggested as a method of treating pre-ovulatory egg binding in a green iguana, Iguana iguana, by Orosz and others (1992). The high probability ofcomplications such as egg peritonitis, as well as anatomical restrictions that result from the chelonian shell, make follicle aspiration unsuitable in most presentations offollicular stasis in chelonian.

Backus and Ramsey (1994) suggested ovariectomy for the treatment offollicular stasis in lizards. Assuming there is no intention ofbreeding, several authors including Backus and Ramsey (1994), Raiti (1995), and Divers (1996), describe ovariectomy/ovariosalpingectomy as the treatment of choice in isolated, anorexic, female lizards. Ovariosalpingectomy in tortoises has been described (Holt 1979; Muller and others 1989; Bennett 1993; Divers 1997). It appears to be a suitable method ofmanaging utero-ovarian disease in chelonians. However it shouldberemembered that there might be potential long-term complications, such as osteoporosis, associated with spaying chelonians. Long-term follow up studies have yet to be performed on chelonians undergoing ovariectomy.

All surgical cases in this series (excluding the single death) showed significant improvement following ovariectomy. All required a prolonged period ofnursing of6-12mo. All cases improved and ate in the spring following surgery. These tortoises were not hibernated the winter after spaying.

• "'lliiiiot!I¥I¥loli,iof¥iiiliri.ittHijii'o',loil;EO'ijl,l#l,t,'grn,'ijI,I;EO'%.:I' Esophagostomy tube feeding was necessary in 1 case for Smo!' Recovery in many cases ofapparent follicular stasis treated by ovariectomy has been prolonged when compared to similar iguana cases. This may relate to the chronic nature ofthese cases.

DISCUSSION

Potential causes offollicular stasis

Absence ofenvironmental cues

4& Environmental cues such as hibernation, photoperiod, and thennoperiod may influence folliculogenesis (Peaker 1969; Licht 1972; Licht 1984; Duvall and others 1982; Bentley 1998). It is possible that an appropriate territory and nesting area must be present before ovulation may occur. Animals that are not pennitted to follow an annual cycle ofchanging photoperiod and changing daily ambient temperatures, may lose their natural breeding cues and fail to ovulate. Jones (1978) gives photoperiod tables appropriate to Testudo spp.

4& In the UK, the weather is unsuitable for most reptiles. Consequently, it is customary to keep tortoises indoors in enclosures with supplementary heat and light, especially during spring and autumn. It is possible that under the influence ofa human waking cycle, breeding cues are deranged.

Loss ofsocial cues

$ Ovulation may be induced, in some way, by the presence of a male tortoise. This could involve pheromones, male courtship behavior such as butting and biting, or the act of mating. Follicular stasis could be the result of a failure of induced ovulation. Regular socialization of females with an appropriate male tortoise might reduce the prevalence of follicular stasis. A similar hypothesis is proposed by Backues and Ramsay (1994) in oviparous lizards. Galbraith (1993) implies that chelonians retain the ability to perform fertile ovulations some time after a successful encounter with a male in some species.

~ Both Jessop (2000) and Wilkinson (2000) believe that some cases offollicular stasis have beenpreviously isolated females experiencing temporary contact with males (e.g., whilst the keeper is on holiday). These cases appear to have gone into follicular stasis when returned into isolation once more.

e Isolated female chelonians might revert to a physiological state requiring induced ovulation, but, following successful contact with a male, may then ovulate unaided for several years once more.

Chronic nutritional disease, systemic illness, and old age

@l Follicular stasis may be a secondary condition, where female tortoises are maintained inappropriately, suffer from any chronic illness, or endocrinopathy.

e Most females with signs consistent with follicular stasis are poorly maintained. Most are kept without adequate heat, photoperiod, balanced nutrition, and with limited exposure to male tortoises. Many may be very old and beyond useful breeding age.

2001 r?roceedings • ~ssociation of Reptilian and ~rnpnibian Reptiles 81 CONCLUSIONS

Follicular stasis appears to be a relatively common condition ofisolated mature female Testudo spp. in the UK. It should be considered onepotential cause ofprolonged anorexia in those mature female tortoises prevented from regular exposure to suitable males.

Often mature female chelonians in a state ofapparent follicular stasis will have failed to pass feces for periods in excess of 3mo. Occasionally, hind limb function will be poor. Typical clinicopathological features may include hypercalcemia, hyperalbuminemia, elevated total protein, moderate anemia, leukopenia, heteropenia, lymphopenia, and elevated alkaline phosphatase. Ultrasonography and endoscopy may reveal the presence of large numbers of well-developed coelomic follicles that fail to either regress or ovulate with time.

Ovariectomy would presently appear to be the most effective fonn of treatment, especially in a nonbreeding situation. An effective medical treatment requires further investigation. Future consideration should be given to the suppression of reproductive function in isolated, captive, females at, or immediately after, puberty.

Chelonians in a state ofprolonged follicular stasis suffer a major metabolic insult. In many cases significant recovery is either unlikely, or at best it will be prolonged. Early intervention would therefore appear beneficial but chronic cases merit a guarded prognosis.

Impaired hind limb function may not improve in the immediate post treatment period. Several cases have remained unable to make voluntary hind limb movements 2-3yr post treatment despite an otherwise good recovery.

Acknowledgments: The author thanks and acknowledges the assistance and data provided by Mike Jessop, MRCVS, ofMountain Ash Veterinary Surgery, UK, and William Lewis, MRCVS, ofSouth Beech Veterinary Surgery, UK.

REFERENCES*

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.1'llliji.'H§j§j.II'('€IIK1-i.iHijii,.I,I."iEU,ijlej§'ii.r.tmmm.jijiil;.01*_:1 Table 1. Clinical data from 10 cases offollicular stasis in Testudo spp.

BenH Tortie R Timmi F MP HGOP Presented 23/09/98 13/07/98 11/08/99 28/07/99 Species Mature Mature Mature Mature Mature F. whitei T. hermanni T.graeca T. hermanni T.graeca Weight 2885g 1880g 2055g 1730g Presenting Prolonged Prolonged anorexia Prolonged anorexia Prolonged Prolonged signs anorexia anorexia anorexia March-Sept May-July May-August Summer 1998 Absence offeces Absence offeces Abs~nce offeces Absence offeces Absence offeces Suspected 01 Suspected 01 Constant aimless obstruction obstruction marching Poor visual Poor visual reflexes Normal vision Poor vision responses Weak hind limbs Weak hind limbs Normal limbs Dragging back legs Others in Death ofin Isolated 30+ yr Isolated 30+ yr Isolated 16 yr Isolated> 24 contact contact Feb months Previous Never laid eggs Never laid eggs Never laid eggs Never laid eggs Unknown reproduction Hematology Borderline anemiaBorderline anemia Borderline anemia Borderline Borderline anemia anemia Leukopenia Normal hematology Leukopenia Panleukopenia Panleukopenia Heteropenia WBC poss depressed Heteropenia Heteropenia Heteropenia Biochemistry Hypercalcemia Hypercalcemia Hypercalcemia Hyperalbuminemia Hyperalbuminemia Hyperalbuminemia Hyperalbuminemia Hyperprotinemia Hyperprotinemia Hyperprotinemia Hyperphosphatemia Hyperphosphatemia Radiology LargeFB NAD NAD NAD NAD Endoscopy Yes Yes Yes Yes No US Follicles present Follicles present Follicles present Follicles present Not possible proligestone proligestone Follicles are Follicles absent post Follicles post >10mm proligestone proligestone Medical Proligestone 1st week Proligestone Proligestone given treatment November 98 22/08, 28/09 and 20/10 20/11/99 Surgical Spayed 05/10/98 Spayed 02/12/98 Spayed 30/11/99 Spayed 25/08/99 Unspayed treatment Liver -lipidotic Liver -lipidotic Liver - lipidotic FB also removed Ovaries sent for histopath Management E-tube4mo E- tube4mo E tube 2mo E tube fitted Environment Peri-operative Peri-operative Peri-operative Peri-operative antibiotics and antibiotics and antibiotics and antibiotics and analgesics analgesics analgesics analgesics Critical Care Critical Care Formula Critical Care Formula Critical Care Formula Critical Care Formula Formula Meds Soloxine None Soloxine Metacam Other Wheels applied Outcome Ate 08/04/99 Ate mid 27/04/99 Ate 28/02/00 Died,....,1 week Moderate improvement (Time post op) 6mo 5mo 3mo N/A Weak hind limbs Appears normal Other Poor vision persisted Viral stomatitis? information

Assimilated from personal data and Lewis 1999, Jessop 2000.

11II. ., • A • • Table 1 continued. Clinical data from 10 cases offollicular stasis in Testudo

TobyR Elizabeth Toots TobyL BigBR Presented 21/10/99 01/09/97 N/A N/A Spurthigh (white's) N/A N/A Species Mature T. graeca mature T. hermanni mature Weight 3620 2125 1880 N/A Presenting Declining appetite (3yr) Oxytocin induced lay, N/A N/A signs 9/96 Anorexia 5mo Prolonged nasal Anorexia Sept/Oct discharge Ophisthotonus. Poor appetite anorexia from 2/99 Owner thought blind Annual male contact 2 Male was present Isolated 40+ yr In contact wks/yr Contact data Isolated 40+ yr stopped 94. (Other died '87) Previous reproduction Never laid eggs Oneegg--90 Never laid eggs Laid eggs last yr Never laid eggs Marked toxic N Hematology Not done heterophils lymphopenia. Reactive lymphocytes Biochemistry Hypercalcemia Hypercalcemia Hypercalcemia Hypercalcemia Hypercalcemia Raised ALT, LDH, Raised urea, Hyperalbuminemia Hyperalbuminemia Amylase creatinine hypouricemia Hyperprotinemia raised ALP Hyperphosphate slight radiodensity in R Slight radiodensity R Radiology ovarian quadrant NAD ovarian quadrant in US Ultrasound inconclusiveNot done Not done Not done Not done Follicles are endoscopic endoscopic endoscopic endoscopic >10mm confirmation confirmation confirmation confirmation Treatment spayed 5/11/99 Spayed 17/3/99 Spayed 30/09/97 Spayed Nov 99 Spayed Liver lipidotic/ fibrotic Liver lipidotic (histo) Liver not biopsied thickened capsule Management Environmental only E- tube fitted pre op E- tube fitted E- tube fitted E- tube fitted Peri-operative Peri-operative antibiotics antibiotics Meds None specified None specified None specified and analgesics and analgesics Recovery seen 2mo Ate -- 10d post op Outcome Ate 8/11/99 Ate 27/4/99 Ate 7/11/97 post op 3days 5-8wk 38 d

Assimilated from personal data and Lewis 1999, Jessop 2000.