Reproductive traits in the spur-thighed tortoise (Testudo graeca terrestris): new tools for the enhancement of reproductive success and survivorship.

The Mediterranean tortoise (Testudo graeca) is listed as "Vulnerable" on the IUCN Red List. Reproductive characteristics and means to increase offspring production were studied in T. graeca terrestris in a semi-natural environment. Courtship and mating occurred during early spring for about 4 weeks, followed by a laying season of approximately 2 months, with a second, shorter mating period in the fall. During the first mating, calcified eggs were already present in the uterus; we inferred that sperm from both mating seasons were stored in the oviduct for fertilization of eggs of the second laying cycle and of the following year. Average egg production was 3.8+/-0.3 eggs/year. Most females laid all of their eggs in a single clutch, but 18% laid in a second clutch, 11-21 days later. X-ray radiography revealed calcified eggs in the uterus about 4 weeks before oviposition. All eggs in the uterus were calcified simultaneously and were laid in a single clutch; if a second clutch developed, those eggs were also calcified simultaneously. Based on endoscopic examinations, ovaries were active throughout the entire year. Plasma progesterone concentrations in females were very low and were detected only soon after oviposition ( 440 +/-141 pg/ml). Plasma estradiol concentrations in females varied from 4.1 +/-1.5 pg/ml to 70.2 +/-29.4 pg/ml, with no clear seasonal pattern. Maintaining tortoises at a low environmental temperature (9 +/-1 degrees C versus 28 +/- 1 degrees C) reduced plasma estradiol concentrations. Giving 2mg/kg tamoxifen (TAM) increased plasma estradiol to 220 +/-33 pg/ml when treatment was given in September but not in late October, winter or spring. Treatment with TAM increased the number of eggs laid during the following laying season to 7.3 +/- 1.0 eggs/year, laid in one to three clutches. In males, plasma testosterone concentrations had a seasonal pattern with the onset of a rise in July from 2 to >4ng/ml, a continued increase to a peak of 12.8+/-5.3 ng/ml during November and a decline thereafter. Artificial incubation in sand at 29 +/-1 degrees C shortened the natural incubation time of 103+/-3.1 days to 83.5 +/- 1.3 days, increased hatching rate from 28 to 53%, and increased survival rate from 51 to 71% at 40 weeks of age. In summary, this study provides options for increasing reproductive performance, hatchability and offspring survival in captive Mediterranean tortoises, and may offer new tools for conservation of animals that are on the verge of extinction.