David C. Rostal

Seasonal Reproductive Cycle of the Desert Tortoise (Gopherus agassizii) in the Eastern Mojave Desert

The seasonal reproductive cycles of male and female desert tortoises (Gopherus agassizii) were studied under semi-natural conditions. Tortoises were maintained in outdoor pens subject to ambient weather conditions and received supplemental food and water. Heparinized blood samples were collected monthly using jugular puncture. Ovarian follicular growth and egg development were monitored using ultrasonography. Mating was observed in the fall (following nesting) and the spring (prior to nesting). Vitellogenesis occurred during the fall prior to hibernation. Nesting was observed from May-early July with females producing one or two clutches. Clutches ranged from 2-7 eggs. Both males and females displayed seasonal testosterone cycles.

EFFECTS OF INCUBATION CONDITIONS ON SEX DETERMINATION, HATCHING SUCCESS, AND GROWTH OF HATCHLING DESERT TORTOISES, GOPHERUS AGASSIZII

Incubation temperature has a direct effect on sex determination of the desert tortoise. Low temperatures (26.0-30.6 C) produce males and high temperatures (32.8-35.3 C) produce females. Pivotal temperature is approximately 31.8 C. Macroscopic and microscopic anatomy of the gonads is similar to that of other turtles. Hatching success and survival is very good between 28.1 and 32.8 C in dry sand (-5000 kPa). Incubation at 35.3 C is lethal for 72% of the eggs and produces weak hatchlings that die within 45 days. Wet sand (-5 kPa) is lethal for desert tortoise eggs. Hatchling size was dependent upon egg size and incubation condition. Hatchlings from eggs incubated at 32.8 and 35.3 C were significantly smaller than hatchlings from eggs incubated at 28.1 and 30.6 C. Hatching mass had no effect on growth rate of hatchlings. Thus, large eggs produced large hatchlings that were larger than their siblings at 120 days of age. Hatchlings from

Non-Lethal Sexing Techniques for Hatchling and Immature Desert Tortoises (Gopherus agassizii)

The development of non-lethal techniques for sexing hatchling and immature desert tortoises is critical to population and ecological studies. Two methods for sexing desert tortoises were evaluated with respect to accuracy, efficiency, and suitability to field application. Laparoscopy was found to be 100% accurate and could be used on hatchlings as small as 28 grams total body mass. Plasma testosterone was 98% accurate for juvenile and immature tortoises ranging from 69190 mm straight carapace length. Plasma testosterone is the most suitable methodology for field studies in that only a small blood sample is required for sexing purposes. Laparoscopy is 100% accurate; however, it may require holding the animal for an extended period of time.

Long-Term Sperm Storage in the Desert Tortoise (Gopherus agassizii)

Many reptilian species exhibit the ability to store sperm in the female reproductive tract for extended periods of time (Gist and Jones, 1987; Birkhead and Moller, 1993). Sperm storage may be used to separate reproductive events such as copulation, fertilization, and hatching to optimize timing of these events (Birkhead and Moller, 1993). The redsided garter snake (Thamnophis sirtalis) ovulates and produces offspring in the spring but mate in the fall (Whittier and Crews, 1986). In this species, sperm from fall matings are thought to overwinter in the oviduct and fertilize ovum the following spring (Crews, 1984; Whittier and Crews, 1986). Reports of delayed egg laying in other species support the hypothesis that stored sperm are viable and used to fertilize subsequent clutches. Sperm storage has been inferred from oviductal flushings and/or observations of sperm in histological preparations of the oviduct (Gist and Jones, 1989; Gist et al., 1990). However, only controlled-mating experiments can demonstrate whether stored sperm are viable. Sperm storage and multiple insemination may play a significant role in turtle reproduction (Gist and Jones, 1989). The ability to store sperm from previous matings and produce viable offspring using these sperm would be necessary for species whose male and female reproductive cycles do not coincide. Discordant cycles have been observed in temperate-zone turtles where time of mating and gamete maturation do not always occur simultaneously (Moll, 1979; Licht et al., 1985). In males of Gopherus agassizii, spermatogenesis begins in early summer and terminates with the onset of fall

Sex differences in plasma corticosterone in desert tortoises, Gopherus agassizii, during the reproductive cycle.

Blood samples from 30 female and 20 male adult desert tortoises, Gopherus agassizii, were collected at monthly intervals during the annual reproductive cycle (April to October). Plasma corticosterone and the sex steroids in each of the samples were analyzed by radioimmunoassay. Mean corticosterone levels in males were significantly higher than in females (P < 0.001) in every month. Male tortoises showed a marked seasonal pattern in plasma corticosterone with a highly significant peak in July, August, September, and October that corresponded with a similar peak in plasma testosterone. Testosterone and corticosterone in the male showed a highly significant correlation (P < 0.0001). The pattern of corticosterone in the female was less marked, with a significant peak in May during the mating and nesting season, but no association with the peak in estradiol in late summer was apparent. The highest levels of corticosterone in the males were associated with the peak in spermatogenesis and intense male-male combat. These results support similar data from other reptiles that suggest increased glucocorticoid secretion during periods of increased activity and metabolism.

Reproductive and Developmental Synchrony in Female Lepidochelys olivacea

The reproductive behavior and movements of arribada nesting olive ridley turtles (Lepido- chelys olivacea) were studied during the 1990 and 1991 nesting seasons (September through November) at Nancite Beach, Santa Rosa National Park, Guanacaste, Costa Rica. Females that normally emerge synchro- nously once a month on Nancite Beach to oviposit delayed oviposition in response to a period of very heavy rainfall. Females retained oviducal eggs for 63 days and emerged synchronously to oviposit at Nan- cite Beach after the rain had ceased. Egg retention in L. olivacea appears to be an adaptation which en- ables delayed oviposition when environmental conditions are unsuitable and may also facilitate reproduc- tive synchrony. We suggest that preovipositional arrest of developing embryos may have occurred while oviducal eggs were retained.

Studies on the Chemistry and Social Significance of Chin Gland Secretions in the Desert Tortoise, Gopherus agassizii

The chemical structure and biological functions of chin gland secretions were examined in a group of captive desert tortoises, Gopherus agassizii, housed at the Desert Tortoise Conservation Center near Las Vegas, Nevada. Monthly measurements of the chin glands of 20 adult males were made over one year. Gland volume varied seasonally, reaching a maximum in late summer. The chin glands of socially dominant males tended to be larger than those of socially subordinate males. Among all males, there was a positive correlation between plasma testosterone levels and size of the chin glands. Behavioral experiments demonstrated that tortoises of both sexes discriminate the chin gland secretions of familiar males from those of unfamiliar males, suggesting that chin gland secretions may function in conspecific recognition. Electrophoretic studies of chin gland secretions revealed the presence of 12-17 protein components ranging in size from 25,000115,000 Daltons. Although banding patterns among males were very similar, slight individual differences existed in the number and size of high molecular weight protein components.

Nest Temperatures and Hatchling Sex Ratios from Loggerhead Turtle Nests Incubated Under Natural Field Conditions in Georgia, United States

Abstract We examined loggerhead nest temperatures and hatchling sex ratios in an effort to more accurately predict hatchling sex ratios produced from 2 barrier islands in the northern management unit (Blackbeard Island National Wildlife Refuge and Wassaw National Wildlife Refuge, Georgia, United States) from 2000 to 2004. Temperature data loggers were placed into 169 nests to monitor incubation temperatures. Average critical period temperatures ranged from 26.3°C to 33.2°C (mean ± SE, 29.2° ± 0.1°C) and indicated seasonal variation in sex ratios. The sex of 669 hatchlings found dead in nests was histologically evaluated (n  =  212 nests; 14–90 nests/yr). The sex ratios varied from 0% to 100% female per nest (n  =  1–53 hatchlings/nest) and average sex ratio for all nests ranged from 55.5% female in 2003 to 85.4% female in 2002. In addition to monitoring nest temperature, 10 hatchlings per nest were euthanized to verify sex during 2003 on Blackbeard Island National Wildlife Refuge (n  =  10 nests) and 2004 on Wassaw National Wildlife Refuge (n  =  9 nests). Sex ratios were analyzed by using an advanced statistical program for evaluating temperature-dependent sex determination and indicated a 1∶1 temperature (temperature that produces a 1∶1 sex ratio) of 28.9°C. We offer an equation for predicting northern management unit hatchling loggerhead sex ratios by using critical period temperature and tested its validity. Sixteen of 18 nests (n  =  10 hatchlings/nest) showed no significant difference between the predicted sex ratios based on the equation vs. sex ratios obtained through histology. Our data indicated that rookery beaches north of Florida are important areas for the production and recruitments of male loggerhead hatchlings into the overall western North Atlantic Ocean and nests deposited earliest within a nesting season are primary contributors of male turtles. We suggest that nest monitoring programs grant such nests particular protection to increase their survivability and the production of hatchlings.

High Pivotal Temperature in the Sex Determination of the Olive Ridley Sea Turtle, Lepidochelys olivacea, from Playa Nancite, Costa Rica

A variety of reptiles possess temperature-dependent sex determination (TSD), including all species of sea turtles examined to date (reviewed byJanzen and Paukstis, 1991; Mrosovsky, 1994). The evolutionary, ecological, and conservational implications of TSD are not clear, but a knowledge of naturally occurring sex ratios is a prerequisite to addressing these issues. In the case of endangered sea turtles, the monitoring of nest temperatures can provide a simple and nonlethal means of estimating hatchling sex ratios. However, the accurate use of nest temperature to estimate sex ratio requires a knowledge of the pivotal temperature (temperature producing a 1:1 sex ratio) and the transitional range of temperature or TRT (range of temperature in which sex ratios shift from all males to all females; Mrosovsky and Pieau, 1991; Mrosovsky 1994). Further, an understanding of how these parameters vary between populations can provide insight into the ecological and evolutionary significance of TSD. Pivotal temperatures and TRTs have been estimated for sea tur-

Seasonal Reproductive Cycle of the Galápagos Tortoise (Geochelone nigra) in Captivity

The reproductive physiology of nine Galapagos tortoises (Geochelone nigra) was studied from February 1988 to May 1989. The study encompassed the annual reproductive cycle to include complete mating and nesting sequences. Male (n = 4) and female (n = 5) seasonal reproductive changes were determined throughout the study with endocrine analysis and ultrasonographic examinations. Males displayed a prenuptial rise in serum testosterone (x― ±SE = 6.62 ± 0.92 ng/ml in August) during which gonadal maturation and spermatogenesis are thought to occur. The male reproductive cycle appears consistent with the prenuptial spermatogenic pattern exhibited by other tropical turtles. In the females, testosterone rose during the mating period (x― ± SE = 499.3 ± 124.6 pg/ml in October) prior to ovulation and is probably related to receptivity in the females. Progesterone was more variable, but also peaked during the mating period (x― ± SE = 1,017.2 ± 220.6 pg/ml in October) and appears related to ovulation. Estradiol rose several months prior to mating (x― ± SE = 75.5 ± 11.9 pg/ml in July) and was correlated with increased serum calcium levels. This increase in estradiol is thought to stimulate vitellogenesis several months prior to mating. Nesting occurred from November 1988 to April 1989, during which six clutches were laid. Clutch size ranged from eight to 17 eggs. Both male and female Galapagos tortoises display seasonal physiological changes that function to regulate annual reproductive patterns. Zoo Biol 17:505–517, 1998.