Captive Breeding Of Four Eye-spotted Turtle, Sacalia Quadriocellata

The Four Eye-spotted Turtle, Sacalia quadriocellata Siebenzock, 1903, has been designated as endangered in the China Red Data Book of Endangered Animals. Recent research indicates that wild populations are decreasing rapidly because of hunting and habitat destruction. With the survival of this species in the wild in question due to increasing threats, gathering biological data to aid the development of sound management programs and creating ex situ assurance colonies of captive specimens is imperative. In 1998, a study on 45 S. quadriocellata was initiated to gather information on the biology of this species and to investigate the feasibility of captive reproduction. In the following six years, there was no courtship behavior in males and no oviposition in females. From 2004 several successful techniques were applied to initiate reproductive behavior and successful reproduction.LHRH-A induce sexual behavior Luteinizing hormone-releasing hormone (LHRH) is known to influence sexual behavior in many vertebrate taxa, but there have been no systematic investigations into the role of LHRH on sexual behavior in any chelonian. We tested the hypotheses that exogenous LHRH analogues would induce male S. quadriocellata to sexual behavior. We examined this by challenging individual males with intramuscular injections of mammalian Luteinizing hormone-releasing hormone anagenue (LHRH-A) in conjunction with human chorionic gonadotropin (hCG), LHRH-A only and hCG only and subsequently exposing them to sexually attractive females for behavior observation. In this study, only exgenous LHRH-A can facilitate, either directly or indirectly, inducing masculine sexual behavior and testicular recrudescence and spermatogenesis on this species. Animals treated with LHRH-A and hCG induce more drastic sexual behavior than LHRH-A only indicates hCG enhances the action of LHRH-A induce the sexual behavior, but hCG alone dose not induce sexual behavior. However, different pharmacological dosage of LHRH-A (0.5ug, 1ug, 2ug per 100g bodyweight) did not correspond to different activity levels. Though the mechanism of LHRH effect was not determined, this study may support that sexual behavior of S. quadriocellata occurs at the beginning of the injection despite the regession of the gonad. This is the first report of exgenous LHRH-A influencing sexual behavior in a chelonian species.Reproductive hormone influence activity rhythm From August 2005 to April 2006, 39 wild S. quadriocellata were divides into 4 groups to study reproductive hormone inducing in freshwater turtles: the first one (6♀,4♂) is low level individuals group; the second one (6♀,4♂) is middle level group; the third one (6♀,4♂) is high level group; the last one (6♀,3♂) is the control group. The cycle of inject is 10 days, hCG and LHRH-A were used in this test, and their behavior was observed in the followed 24 hours after injecting. Effects of activity rhythm induced by LHRH are as follow. There was no difference in dial activity rhythm and has significant difference in year activity rhythm between test group and control group. The males in the three injecting hormone group behaved obvious courtship and copulation behavior, while the control group haven't see any courtship behavior. The resting behavior, moving behavior, eating behavior and other behavior of male turtles were no difference in groups. The resting behavior, moving behavior, eating behavior of female turtles were changed significant difference between the injected groups and the control group. It has a combine to reproductive invest. Low level dosage cause more intense courtship, too high level dosage suppress function of LHRH; It assumed forward that two reasons maybe cause reproductive obstacle. First, have no adaptive environment factors that MLT secreted too much in reproductive period suppress LHRH secreting. The second reason is that capture stress acting on hypothalamic-pituitary- adrnal axis (HPA) cause gucocorticoids secreted too much that suppress testosterone.Follicular development cycle Ultrasound investigations on ovarian activity and follicular growth were performed on 8 adult female S. quadriocellata in captive from January 2007 to May 2008. The results showed: The number and size of Four Eye–spotted turtles'growth of ovarian follicles were in obvious cyclical changes. Vitellogenic follicles ranged between 4～24mm (n=186). At August, the number of follicles began to increase along with the temperature decreased, to December the number of preovulatory follicles was the largest and the ovary was mature. Size of preovulatory follicles ranged between 19～24mm (n=56) and the largest was detected in December. Follicles reached ovulatory size from January to March. Atretic follicles ranged between 10～20mm (n=14) were only detected from October to the following March.Reproductive hormone influence follicular development From August 2004 to December 2004, 12 S. quadriocellata were divide into 3 groups. LHRH-A and HCG were used in this test, ultrasound and X-ray were used to exam the development of these eggs. Some females from both of two injecting hormone groups had eggs and laid zygotes, while there is no one had eggs in the control group. The author assumed forward that maybe the lack of LHRH is the main reason to make animal can not reproduce. In the second expriment, 18 female S. quadriocellata were divided into 3 groups, 6 individuals each group: The first group is high level, injected with LHRH-A 0.5ug/100g bodyweight, HCG 200IU every individuals. The second group is low level, with LHRH-A 8ug/100g bodyweight, HCG 200IU every individuals.In noverber, 2005, the average number of follicles in three group were significant different (χ2=11.645, df=2, P=0.003). The high level was 0.67 per side, low level was 2.08 per side, and the control was 1.08 per side. The average number of follicles in three group were no significant different (Mann-Whitney U=1777.5, P=0.856). The results indicates that inject LHRH-A to female S. quadriocellata in reproductive season can induce follicles development, the influence was lie to the dosage.Nesting and incubation Ovulation occurred between December and March, and no significant correlation was found between ovulation and food intake. Females laid a single clutch every year, with an average clutch size of 2.47 eggs (n=34, range=1-4). A total of 84 eggs were laid, of which 13 were damaged, 52 were undamaged but infertile, and 19 were undamaged and fertile. Of the fertile eggs, nine hatched with a mean incubation period of 105.9 days (n=9, range=89-122 days) as the incubation temperature ranging between 24 and 27°C. To our knowledge, the current study is the first report to successful breeding of this species in captivity.Eco-breeding The results indicate that the stress of captivity contributed to the lack of reproductive hormones, directly leading to the stagnation of the gonadal development and suppression mating behavior in S. quadriocellata. From January 2007 to December 2009, an ecological husbandry brought forward in terms of captive environment, photoperiod and nutrition in this paper. The results show that: of which follicle development never occurred, began to ovulate when placed in the outdoor pond that reduced captive stress. Induvuals feed with admixture feed layed more eggs than others. Shorten the photoperiod induced the follicular development than prolong. The experiences reported here are helpful for breeding of other species.