人工养殖的幼年绿海龟甲状腺结构与功能的研究

夏中荣 李丕鹏 古河祥

(1.广东惠东海龟国家级自然保护区管理局,惠东,516359;2.沈阳师范大学两栖爬行动物多样性研究课题组,沈阳,110034)

1 Introduction

There is a large body of evidence showing that thyroid hormones play an important role in the regulation of development，growth and reproduction in fish[1-2]；In reptiles thyroid physiology influences many processes such as shedding，growth，development，reproduction，metabolic rate，nutrient assimilation and activity[3].Preliminary data suggest higher Deiodinase activity in red-eared sliders than in snapping turtles，suggesting that the Deiodinase enzymes(DIs)may be a hypothetical metabolic pathway for reductive debromination in red-eared sliders[4-5].Triiodothyronine(T3)and tetraiodothyronine(thyroxine or T4)are protein bound and distributed in the bloodstream to the peripheral tissues.T4is converted to the more active form，T3，via de-iodinases，but in reptiles T4predominates[3].Turtles that showed persistent deficits in feeding，activity or both during recovery had significantly lower free T4than did turtles with no such deficits[6].Studies on the seasonal change of serum or plasma thyroid hormone level in lizards and snakes have shown a link to environmental temperature change，with a peak of T4being in summer months and a trough in winter months when the environmental temperature drops[7-10].These studies imply that plasma T4may support energy consuming activities，such as nutrient assimilation，activity，and reproduction.Plasma T4in green turtles decreased with deprivation of food，but did not do so in Kemp’s Ridleys.Increased feeding did not affect circulating levels of plasma T4in either species.Plasma T3，in contrast，tended to decrease during fasting and increase with refeeding or increased feeding[11].

It has been suggested that thyroid hormones therefore should exhibit distinct annual cycles in temperate-zone animals，including turtles，during periods of metabolic activity[12].However，previous studies have been mostly limited to the description of hormone levels.Licht et al.[8]reported seasonal changes in serum thyroxine levels in captive male green turtle(Cheloniamydas)，Another study found sexual dimorphism in the annual cycle of thyroxine in adult Kemp’s Ridley turtles(Lepidochelyskempii)，but not in immature male and female loggerhead sea turtles and green turtles(C.mydas，Carettacaretta)[10].Some other studies showed that thyroid status had important influence on growth of young turtles(Trachemysscriptaelegans)[5].In addition，metabolism and the growth of young turtles are closely related to plasma T4concentration[8].

Although many experiments have been conducted to describe thyroid hormones and their relationship to external temperature and metabolism in squamates，and recently in tortoises and soft-shelled turtles[13-16]，the function of thyroid hormones in other reptilian species has rarely been investigated[10].

As an important animal group，sea turtles are studied to understand the function of thyroid hormones.Useful conservation strategies of the globally endangered sea turtles are proposed based on improved understanding of its basic biology.The study of thyroid hormones in sea turtles would enrich the knowledge of their basic metabolic physiology，growth and development，and thus can contribute to the basis of conservation strategies and management[10].

Furthermore，research and conservation ofC.mydashave been conducted consistently for several years at the National Gangkou Sea Turtle Reserve(NGSTR).A major focus of the program is head-starting hatchlings from NGSTR for one to two years before releasing them to restock the wild population.This program mainly aims to raise the survival rate of the sea turtles，since the juvenile turtles are particularly vulnerable to their natural enemies in wild，with average survival rate less than 1% for the first one to two years[17].The program provides a feasible arena for future research of sea turtles in view of the inherent difficulties in working with wild turtles[10，18-19].

The objective of the present study is to describe the structural changes of thyroid gland and the variation in serum thyroid hormone levels in juvenile green turtles of 8 to 20 month-old.The relationship of thyroid hormone levels with the dynamic metabolic activation induced by temperature，feeding or other activities[10]，as well as the growth pattern of captive-reared green turtles would also be investigated.The study period lasted from 2006 to 2008.Monitoring of thyroid hormones may be useful for triage，monitoring of recovery，and assessing readiness for sea turtles release in future.

2 Materials and Methods

2.1 The place of the study

The National Gangkou Sea Turtle Reserve(NGSTR)(N22°33’，E114°54’)in Guangdong，China is the first and only National Reserve to protect the endangered sea turtles in the mainland China.The NGSTR has a primary and important nesting beach of green turtles in southern China.At the NGSTR，rehabilitation of sea turtles from stranding or confiscation incidents is in place.Recovered individuals are released to the wild.The NSGTR is also devoted to conducting public education on conservation of sea turtles and marine resources.

2.2 Artificial incubation and captive rearing

One hundred juvenile green turtles were hatched from nesting turtles at the NGSTR in August，2005，and three female adult turtles were captive-reared as hatchlings from the NGSTR in the Sea Turtle House of the NGSTR.The turtles were fed with marine fish，shrimp，compound feed，leafy vegetables，and giant kelp(Thalluslaminariae).Daily feeding was about 5% of the body weight of the turtles.We recorded the data including daily air temperature，water temperature，and turtles’ response in feeding and activities.

2.3 Morphometry

Body weight were taken with an electronic scale(accuracy of 0.1 g)，curved carapace length(CCL)with digital electronic calipers(GUI-03000002，precision of 0.02 mm)，vernier calipers(accuracy of 0.01 mm)and soft tape(accuracy of 0.1 mm)，respectively.From April 2006 to May 2007，on the 20thof each month，we randomly sampled and surveyed the body weight and CCL of 9 to 16 green turtles hatched in 2005.

2.4 Observation of thyroid at microstructure level

10 individuals of juvenile were chosen randomly sampled once every 3 or 4 months from March to December each year from 2006 to 2007.10-15 slices of thyroid tissue were collected from each individual.The fixed thyroids were conventionally embedded in paraffin，sectioned at 7 μm thick slices，and stained using a hematoxylin/eosin procedure[20].We observed thyroid follicular shape，cell size，shape and height and the secretion of particles，etc.at the microstructure level using OLYMPUS BH-2 microscope，LEICA DFC 280 microscopic imaging system，and camera software：LEICA IM50 Version 1.20 Release 15.

2.5 Observation of thyroid at ultrastructure level

From March to December each year from 2006 to 2007，10 juvenile individuals were chosen randomly for ultrastructural study.11-25 slices of each individual were made.We rapidly sampled thyroids of turtles，cut into 1 mm3tissues，fixed in 4% glutaraldehyde under 4℃ and then in 1% osmium acid，embedded with Epon812，electronically sectioned at 80 nm，and doubly stained with two-oxygen uranium acetate-lead citrate.Under PHILIPS TECNAI-10 TEM(Philips TEM companies in the Netherlands)，we observed sea turtles thyroid cell secretion granules，mitochondria，RER，and other cell ultrastructural changes，and photographed them with Morada Soft Imaging System.

2.6 Determination of thyroid hormone tests

In 2007，on the same day of each month before feeding(on the 20th，at 9：00 am)，blood samples were taken from 5 juveniles selected from 60 healthy green turtles hatched in 2005，1-2 mL blood taken from the carotid sinus with a vacutainer，were centrifuged at 3500 g/m for 5 min，and the serum samples were frozen and stored at under-40℃ for the subsequent analysis.Thyroid hormone levels were measured with a chemiluminescent analyzer(Beckman/Coulter Access immunoassay system).Annual hormonal changes were analyzed.

Using the software SPSS 11.5，we calculated the average body weight，CCL，T3and T4concentrations，mapped the growth curves and concentrations of thyroid hormone in the different months.

Tab.1 Body weight and growth curve carapace of 8 to 20-month-old green turtles born in 2005

3 Results

3.1 The green turtle growth

Data on body weight and curved carapace length(CCL)of green turtles born in 2005 was shown in Table 1.From 8-months to 20-months，body weight rose from(286.7±34.6)g to(1203.5±110.5)g，and the curved carapace length increased from(125.2±5.1)mm to(215.6±7.2)mm.Growth rates varied in different months and peaked in September(Fig.1).Average weight gained per month reached a maximum at 256.7 g，and for CCL，an increase of 25.2 mm per month was observed.However，from January to April，body weight decreased instead，corresponding with a small decrease in CCL.

3.2 The general structure of green turtle thyroid

Unlike the Chinese alligator(Alligatorsinensis)which had a pair of thyroids beside the trachea[21]，the green turtle had a single thyroid，right above the bifurcation of the artery arch，and the junction of the right carotid artery.This gland was covered with a thin layer of connective tissue，and a great deal of small blood vessels and rich capillaries.The thyroid gland organ was composed of many follicles between which were some sparse connective tissues and capillaries.Many follicles were round or oval，follicular epithelial cells as the single layer of thyroid cells，due to different activity periods，there were of high cylindrical or cubic cells(Fig.2).Follicles were filled with eosinophilic colloid，depending on the functional active status，there were swallowing bubbles between colloid and thyroid cells.

3.3 Changes of green turtles thyroid structure

Observed microstructure of the thyroid throughout the year，showed that height of thyroid cells was the lowest in December.These cells were almost cube-shaped，with a ratio of height to width was about 1：1，indicating these cells were inactive at this time，colloid was relatively thin in these follicles，slightly stained and without swallowing bubbles；In March，the thyroid cells were higher，the cells were low columnar，and the proportion of the nucleus to the cytoplasm was larger，the ratio of cell height to width was about 2∶1；However，in July，the thyroid cells were significantly higher than March，and the ratio of nucleus to the cytoplasm increased，indicating the high cylindrical thyroid cells were active.Ratio of cell height to width was about 3∶1 or even higher.The colloid in the follicles was rich and stained deeply.Numerous swallowing bubbles of varying sizes appeared between epithelial cells and colloid(Fig.3).

Electron microscopic observations further showed that thyroid cells at different heights had significantly different levels of secretory activities，the variety and number of organelles involved in secretory activities was also different.In the cytoplasm of cells during the period of inactivity，there were few developed organelles，such as mitochondria and Golgi bodies were less or not significant.Secretory granules were concentrated in the free end of the follicular epithelial cells(Fig.4).The high cylindrical thyroid cells were rich in mitochondria and developed Golgi bodies.Different sizes of secretory granules were scattered in the cytoplasm(Fig.5)，showing that thyroid cells were in active metabolic state.

3.4 Annual changes in thyroid hormone of the green turtle

Data of the average air temperature，water temperature，and the average concentration of serum T3，T4value(n=3-5)across the twelve months was shown in Fig.6.The lowest air temperature and water temperature appeared in January，while the highest values were in July.The highest value of serum T3also happened to appear in July，but the highest value of the serum T4was in October.The values of T3and T4were significantly different across months(P<0.01，2-tailed).By the Pearson correlation test，T3and the air temperature，water temperature has a very significant positive correlation(Fig.6)(P<0.01，2-tailed).

4 Discussions

In reptiles，the thyroid plays important roles in facilitating reproduction[22-23]，increasing metabolic rate[24-25]，promoting molting[26]and so on.Many factors also influence thyroid values including age，sex，temperature，diurnal changes，seasonal changes，day length，shedding，illness，stress and breeding[3].

4.1 Relationship between growth rate of green turtles and the environmental temperature

The growth of the green turtle baby kept in tub maintenance was greatly influenced by the amount and quality of the available food and on the condition of water temperature[27-28].Findings of this study showed that the average annual air temperature was about 23.9 ℃ and water temperature was 23.0 ℃.Green turtles in the captive facility were active almost year round and there were few phenomena of hibernation at NGSTR.When water temperature dropped to 15 ℃，juvenile turtles still ate，but food intake was significantly reduced[19].Occasionally when the temperature dropped to 10 ℃，the juvenile turtles stopped eating and showed brief dormancy by floating on the water surface.Growth rates varied in different months’ temperature and peaked in September(Fig.1).

Green turtles are ectothermic reptiles.Therefore，the external temperature greatly impacts its metabolism，growth，development，and other biological or physiological activities.As mentioned before，the highest temperature(air temperature and water temperature)was in July，but the fastest growth rate of green turtles was in September.Turtles consumed excessive energy in the high temperature or，excessive thyroid hormone possibly speeded up the process of protein decomposition.

4.2 Influence of serum T3，T4 concentration on the growth rate

The green turtle serum T3level peaked in July，yet the fastest growth rate was in September，lagging two months later.This lag is possibly due to the high temperature in July decreasing the activity of some enzymes.Continuous exposure to light inTestudohorsfieldicaused thyroid production to peak at five days with a gradual decline to inactivity by 35 days(Lynn，1970)[9].Highest value of serum T4concentration was in October，and high growth rate extended to December.Findings presented in Fig.6 imply that secretion of thyroid hormone at the beginning was very slow，but last in long duration.Because thyroid hormone secretion is naturally chronic and continues to appear in the circulatory system，its effect is more lasting[29].However，Plasma T4levels in captive green sea turtles，with little environmental annual and seasonal temperature variation(Cayman Turtle Farm，Grand Cayman Islands)remained uniform[3，8].This is due to the difference in latitude between the two places.

4.3 Relations of ambient temperature and serum thyroid hormone concentration of green turtles

Annual ambient temperature(air temperature，water temperature)and T3，T4serum level changes across time were similar to a parallel single-peak curve.The highest peak of serum T3and the ambient temperature were in July.This is consistent with the findings of thyroid hormone concentration increasing with high temperature in cold blooded vertebrates[29].Moon et al.[10]reported that temperature drives the seasonal cycle in plasma thyroid hormone levels.Wild immature green turtles also exhibited a tendency for T4increase as water temperature increased，but captive sea turtles exhibited higher plasma thyroxine levels than the wild ones.This difference may be ascribed to nutritional effects[10].

With the temperature lower，thyroid hormone levels drop，indicating slower metabolism in cold winter[11].Wild animals with reduced thyroid activity were considered to be adapting to inadequate food sources in the winter.In order to tide over the winter，animals have to be on a diet，by adjusting the thyroid activity level，lowering metabolism to adapt themselves to reduced nutrition and energy intake，and delay the processing of energy material consumption[30].In semi-captive males and female Kemp’s Ridley sea turtles(Lepidocheliskempi)thyroxin levels also displayed seasonal changes that coincided with changes in reproductive behaviour and physiology[31].

Cyclical and phased thyroid hormone release is controlled by the hypothalamus-pituitary-thyroid axis.Temperature and sunlight are also involved in the regulation of the cycle，particularly in cold-blooded animals[29].The green turtles exuberant feeding period was in autumn[19]，possibly because of optimal factors such as temperature and light stimulation in autumn.This resulted in higher levels of thyroid hormone secretion as observed in this study，and an increase in appetite and food intake，which was conducive to the physical storage of enough nutrients to ensure them survive through the winter.

Wu et al.[32]reported that there was an important relationship between the snake cobra energy metabolism and thyroxine(T4)，and Triiodothyronine(T3)during hibernation.Before hibernation，there was a low level of serum T3，T4involved in the adjustment process of declining the metabolic rate.Low temperature limited the secretion of thyroid hormones during hibernation.When the temperature rose，the serum hormones significantly increased and played a promoting role in metabolism，so that these animals went into a post-hibernating period.Therefore，the temperature of the cobras’ living environment and its T3，T4concentration level had a very significant positive correlation[32].Level of T4in the desert tortoises(Gopherusagassizii)also showed significant cyclical changes：a minimum during hibernation and an increase during post-hibernation[13].Although green turtles did not hibernate in the NGSTR，annual thyroid hormone changes were virtually the same as that of cobras，and other reptiles.This may be because the endocrine system of these three reptiles was conserved in long-term evolution.

4.4 Relations of T3 and T4 concentration in serum of green turtles

Thyroid hormone secreted into the blood was mainly T4，which accounts for about 90 percent of the total.This was consistent with Thomas H.Boyer’s finding[3]：T4predominates in reptiles.A small number of T4lose an iodine ion to change into T3.Therefore only a small amount of T3occurred in the blood，but the effectiveness of T3in promoting growth was three to five times more than that of T4[23].From June to September in our experiment，green turtle serum T3was significantly higher than T4concentration，accounting for more than 80% of total serum thyroxine.Especially in July，the serum T3was 99.2 percent of the total thyroxine，while serum thyroid hormone was mainly T4in the remaining month，more than 83.1 percent of the total thyroid hormone.The cause of this phenomenon is unknown and deserved further study.It may be related to thyroxine binding protein(TBP)，TBP was minimal upon emergence from hibernation and maximal in late summer，about a month after the peak in plasma T4[3，33].

In short，temperature was most likely an external factor affecting the growth rate of green turtles，and thyroid hormone was an internal factor regulating turtle growth.Ambient temperature，which fluctuated with the season，affected thyroid hormone level and in turn affected sea turtle growth.These created a pattern of green turtle growth throughout the year.

4.5 Structure and changes of the green turtle thyroids

Chelonians and snakes have single thyroid glands，lizards and crocodilians have single，paired or lobed thyroid glands[3].The structural change of reptile thyroid tissue was mainly correlated with seasonal temperature and reproductive status[34].For example，the bat’s thyroid showed marked seasonal variation in weight，quantity of colloid and follicular epithelial height[35].The research of two reptilian species of vipers showed that the height of thyroid epithelial cells was the lowest in winter，reached the highest post-hibernation，decreased in the summer，and reached the second peak in fall[36].However，natricine was different in the two snakes.The height of thyroid cells reached to highest in February.Studies on cobras also showed that the thyroid columnar epithelium cells had a similar peak of annual cycle changes.Peak height of the cells was in May，at its peak before the thyroid hormone levels.They believed that there was a significant positive correlation between the height of thyroid cells and activities of cobras[9].However，studies of cobras performed by Chiu et al.[37]showed that thyroid epithelium cells reached maximum height in May，October and November；lowest in August.They believed that the thyroid function may be related to animals’ activities，but does not matter with temperature.This may indicate that cobras and sea turtles thyroid work differently.

In a wide variety of temperate reptiles(Sceloporus，Lacerta，Dipsosaurus，Natrix，Vipera，Gopherus，Chrysemys，Trachemys，Pseudemys)thyroid activity is high during summer higher temperatures and low during winter lower temperatures[3，13，15].In this study，with the increase of ambient temperature，the microstructures and ultrastructure of the thyroid gland of green turtle changed obviously，and its function changed from inactive to active secretory state.

5 Conclusion

By observing the sea turtles’ thyroid structure and its changes，we showed that not only did the green turtle thyroid have similar structure to other reptile thyroids，but also the microstructure and ultrastructure of juvenile green turtles thyroid also showed obvious annual changes.These changes were basically consistent with the annual change of thyroid hormone concentration in blood，which further indicated that the function of thyroid and thyroid hormone level in juvenile green turtles were mainly related to their active state.

Acknowledgements：Our work is permitted by the Guangdong Huidong Sea Turtle National Nature Reserve.We greatly appreciate the assistance of staff from the Sea Turtle Nature Reserve，and express sincerely thanks to Dr.Cheng I-Jiunn from National Taiwan Ocean University，Fong Jonathan and Vicky Zhuang from University of California，Berkeley all of they who provided us valuable insight and guidance during our study.We also thanked Ms.Connie Ka-yan NG of the City University of Hong Kong to proof read this manuscript.