Male Turtles Research Articles

Estimation of Flattened Musk Turtle (Sternotherus depressus) Survival, Recapture, and Recovery Rate during and after a Disease Outbreak

We estimated survivorship, recapture probabilities and recovery rates in a threatened population of Flattened Musk Turtles (Sternotherus depressus) through a disease outbreak in Alabama in 1985. We evaluated a set of models for the demographic effects of disease by analyzing recaptures and recoveries simultaneously. Multiple-model inference suggested survival was temporally dynamic, whereas recapture probability was sex- and age-specifc. Biweekly survivorship declined from 98–99% before to 82–88% during the outbreak. Live recapture was twice as likely for male turtles relative to juveniles or females, whereas dead recoveries varied only slightly by sex and age. Our results suggest modest reduction in survival over a relatively short time period may severely affect population status.

Peptide Hormones Versus Steroid Hormones: Case Studies from Snail and Turtle Populations

The proposed mechanisms of masculinization in gastropod mollusks (imposex) and feminization in oviparous vertebrates has focused primarily on the interaction of xenobiotics with the steroid hormone system. The evidence does not support the proposed mechanism of imposex induction, inhibition of CYP 19 (aromatase), since the changes in steroid titers occur only in the later stages of imposex. In addition, the role of vertebrate sex steroid hormones is limited in gastropods. Recent evidence suggests that peptide hormones play a key role in masculinization in snails and may be a more plausible mechanism of imposex induction. Steroid hormones play a role in feminization of oviparous vertebrates. Vitellogenin (Vtg, egg yolk) induction has been used extensively as a biomarker of feminization. However, in some species of turtles there is evidence that high levels of estradiol are necessary to initially induce Vtg. Once this imprinting occurs, lower levels of estradiol can then induce high levels of Vtg. It was found that environmental levels of estradiol do not always induce Vtg in male and juvenile turtles. However, these same levels of estradiol significantly elevate female Vtg levels. Elevated female Vtg may lead to larger, but fewer, eggs, and therefore fewer offspring. However, these offspring may have better survivorship. Alternatively, elevated Vtg may lead to a larger number of smaller eggs, which have been shown to produce smaller hatchlings with reduced survival. The effects of elevate female Vtg on fitness are difficult to assess in such long-lived species.

Vitellogenin induction in painted turtle, Chrysemys picta, as a biomarker of exposure to environmental levels of estradiol

Ponds within cattle farms often support turtle and fish populations and are impacted by manure runoff. Cattle excrete metabolized (glucuronide-conjugated) hormones in feces and urine into these ponds, and bacteria cleave the glucuronide metabolites to active steroids, which can be stable for several weeks in wastewater. The objectives of this study were to (1) assess levels of xenoestrogens found in ponds near livestock pastures; and (2) assess whether these levels of xenoestrogens induce vitellogenin (VTG) in painted turtles in the laboratory and field. We collected water twice, 6 weeks apart, and placed turtle traps weekly into two ponds, which receive runoff from beef cattle pastures, and into one pond with no cattle farm effluents. Water E 2 levels were analyzed using C 18 solid phase extraction disks and detected in a radioimmunoassay (RIA). Plasma was collected from painted turtles ( Chrysemys picta) captured from these ponds and VTG levels were measured via enzyme linked immunosorbent assay (ELISA). Nine additional turtles were collected from a pond at the South Carolina Botanical Gardens, which receives no farm runoff, and were exposed in the laboratory to nominal concentrations of 0.15, 1.5, and 15 ng/l estradiol (static renewal) over a 28-day period, followed by 14 days in clean water. Plasma samples were taken weekly for VTG measurement via ELISA. Levels of free estradiol in the water column of farm ponds range from 0.05 to 1.80 ng/l, as measured by RIA, and up to 7.4 ng/l as measured by ER-β binding affinity. This is similar to what has been reported in streams receiving sewage treatment works (STW) effluents. In the laboratory, plasma VTG in male painted turtles could not be induced even at the high E 2 dose (9.45 ng/l) after 28 days. In the field, VTG levels were induced only in females when compared with animals from the SC Botanical Gardens. Adult male turtles need to be primed with high doses of E 2 prior to being able to respond to exogenous E 2. Given that males would not typically be sensitized in the wild, environmentally relevant levels of E 2 may not be sufficient to affect them. However, higher VTG levels in females could potentially change their reproductive fitness by altering egg size or by shifting energy allocations away from other survival needs. Long-term studies are needed to study potential impacts of VTG induction on female turtle reproductive success.

Modulation of the adrenocortical stress response in marine turtles (Cheloniidae): evidence for a hormonal tactic maximizing maternal reproductive investment

AbstractThe relationships between reproductive condition, level of reproductive investment and adrenocortical modulation to capture stress in marine turtles form the basis of this study. When subjected to either capture or ecological stressors, nesting marine turtles have demonstrated adrenocortical responses that are both small in magnitude, and slow in responsiveness. These observations were further investigated to determine whether this minimal stress response was a physiological strategy to maximize reproductive investment in adult green Chelonia mydas and hawksbill Eretmochelys imbricata turtles. Female green and hawksbill turtles exhibited a decrease in adrenocortical responsiveness with progressive reproductive condition. Breeding turtles exhibited most suppression of their adrenocortical response to capture compared to both non‐breeding and pre‐breeding female counterparts. Nesting green turtles maintained a suppressed adrenocortical response to capture throughout the nesting season despite decreased reproductive investment. In contrast, male green and hawksbill turtles were less able to modulate their corticosterone (B) response to acute capture stress. During breeding, male turtles possessed significantly greater adrenocortical responses to capture than females. These results could indicate that the large reproductive investment necessary for female marine turtle reproduction might underlie the marked decrease in adrenocortical responsiveness. This hormonal mechanism could function as one strategy by which female marine turtles maximize their current reproductive event, even though under certain situations this mechanism could entail costs to female survival.

Polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), biphenyls (PCBs), and organochlorine pesticides in yellow-blotched map turtle from the Pascagoula River basin, Mississippi, USA.

Concentrations of polychlorinated-dibenzo-p-dioxins (PCDDs), -dibenzofurans (PCDFs), -biphenyls (PCBs), and organochlorine pesticides were measured in tissues of map turtles collected from two locations in the Pascagoula River drainage of Mississippi, USA. PCBs were most predominant among the organochlorines with a concentration of up to 99 ng/g, wet weight (580 ng/g, lipid weight) in livers. The greatest concentration of PCDDs/DFs of 1.1 pg/g, wet weight (15.76 pg/g, lipid weight) was found in the liver of a male turtle. The measured concentrations of organochlorines were less than those reported for turtles from the Great Lakes Basin and upper St. Lawrence River. PCBs contributed 90-99% of the total estimated 2, 3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TEQs). Particularly, PCB congeners 105, 118, and 156 accounted for 68-80% of the estimated toxic potency of PCBs in turtles.

Embryonic treatment with xenobiotics disrupts steroid hormone profiles in hatchling red-eared slider turtles (Trachemys scripta elegans).

Many compounds in the environment capable of acting as endocrine disruptors have been assayed for their developmental effects on morphogenesis; however, few studies have addressed how such xenobiotics affect physiology. In the current study we examine the effects of three endocrine-disrupting compounds, chlordane, trans-nonachlor, and the polychlorinated biphenyl (PCB) mixture Aroclor 1242, on the steroid hormone concentrations of red-eared slider turtle (Trachemys scripta elegans) hatchlings treated in ovo. Basal steroid concentrations and steroid concentrations in response to follicle-stimulating hormone were examined in both male and female turtles treated with each of the three compounds. Treated male turtles exposed to Aroclor 1242 or chlordane exhibited significantly lower testosterone concentrations than controls, whereas chlordane-treated females had significantly lower progesterone, testosterone, and 5[alpha]-dihydrotestosterone concentrations relative to controls. The effects of these endocrine disruptors extend beyond embryonic development, altering sex-steroid physiology in exposed animals.

Interactions between Behavior and Plasma Steroids within the Scramble Mating System of the Promiscuous Green Turtle, Chelonia mydas

We measured plasma androgen (combined testosterone and 5α-dihydrotestosterone) (A) and corticosterone (B) in the promiscuous green turtle (Chelonia mydas) during courtship in the southern Great Barrier Reef. This study examined if reproductive behaviors and intermale aggression induced behavioral androgen and adrenocortical responses in reproductively active male and female green turtles. Associations between reproductive behavior and plasma steroids were investigated in green turtles across the population and within individuals. Levels across a range of both asocial and social behaviors were compared including (a) free swimming behavior; (b) initial courtship interactions; (c) mounted behavior (male and female turtles involved in copulatory activities); (d) intermale aggression (rival males that physically competed with another male turtle or mounted males recipient to these aggressive interactions); and (e) extensive courtship damage (male turtles that had accumulated excessive courtship damage from rival males). Behavioral androgen responses were detected in male turtles, in that plasma A was observed to increase with both attendant and mounted behavior. Male turtles who had been subjected to intermale aggression or who had accumulated severe courtship damage exhibited significantly lower plasma A than their respective controls. No pronounced adrenocortical response was observed after either intermale aggression or accumulation of extensive courtship damage. Female turtles exhibited a significant increase in plasma B during swimming versus mounted behavior, but no change in plasma A. We discuss our results in terms of how scramble polygamy might influence behavioral androgen interactions differently from more typical combative and territorial forms of male polygamy.

Seasonal Changes in Gonadal Activity and the Effects of Stress on Reproductive Hormones in the Common Snapping Turtle,Chelydra serpentina

The seasonal gonadal cycle (including gonadal histology, sex steroids, and gonadotropins) was studied in freshly captured common snapping turtles,Chelydra serpentina,from Wisconsin, and the effects of capture stress were evaluated. The ovarian and testicular cycles are shorter than those reported in other freshwater turtles; the cycles commence in mid-May and terminate in early September, immediately after the completion of gonadal growth and maturation. In the female, testosterone (T), 17β-estradiol (E2), and progesterone (Pro) were highly correlated with follicular growth and vitellogenesis. Ovulation in captivity and under natural conditions occurred after mid-May. In captivity, ovulation was a rapid process (24–48 hr); as the follicles descended into the uterine horns there was a significant increase in E2and Pro and eggs were retained in the uterine horns for about 2 weeks before oviposition. In the male, T was sig nificantly correlated with testicular growth and spermiation. Follicle-stimulating hormone (FSH) reached significantly higher levels (P<0.01) in males (8.99±0.38 ng/ml) than in females (2.66±0.22 ng/ml), but luteinizing hormone was undetectable in both sexes. FSH was not correlated with the steroids in either sex. Sex steroids and FSH began to rise before spermiation and vitellogenesis and remained elevated until completion of gonadal growth and maturation. Leydig cells, the main source of plasma androgen in this species, became active shortly after emergence from hibernation and remained steroidogenically active for the rest of the cycle. Sertoli cells became active only after spermatogenesis was under way but also stayed active for the rest of the summer. Courtship and mating behaviors were observed in spring, summer, and fall. The snapping turtle is strictly aquatic with no basking behavior and limited behavioral thermoregulation so there is little daily fluctuation in body temperature. Environmental correlates indicate that the snapping turtle is temperature dependent: recrudesence occurs with a slight increase in water temperature during spring and early summer, while a dramatic drop in gonadal activity accompanies a slight decrease in temperature in fall. Changes in temperature may underlie changes in gonadal activity in the face of relatively stable FSH. Male turtles subjected to captivity and periodic blood sampling show a significant decline in T. The hormonal levels continued to decline whether the turtles are exposed to optimum or extreme temperatures. However, there is more rapid decline in T values in animals with regressed testes (June) than in those with well-developed testes (July). Male and female turtles kept in captivity at different phases of the cycle exhibit different patterns and degrees of response to stress, possibly related to the hormonal levels and the condition of the gonads.

Synergistic Responses of Steroidal Estrogensin Vitro(Yeast) andin Vivo(Turtles)

Many environmental agents exert estrogenic activity. Previous studies from our laboratories demonstrated that certain combinations of environmental estrogens (i) reverse the sex of male turtle embryos in a synergistic manner (Bergeron et al., (1994)Environ. Hlth Perspect.102, 780–782), and (ii) synergistically transactivate the human estrogen receptor (hER) in yeast and mammalian cells (Arnold et al., (1996)Science272, 1489–1492). Because our findings with synthetic estrogens suggested that combinations of naturally-occurring steroidal estrogens might also produce synergistic activity of the ER, we used the same model systems to measure the activity of combinations of steroidal estrogens. The activity of combinations of estrone, estradiol-17β or estradiol-17α in yeast strains expressing hER was synergistic at submaximal concentrations of both estrogenic compounds. However, synergy was not observed with mixtures of estrogens when the concentration of one of the estrogens alone was maximally active in yeast. Ligand-binding assays in yeast performed with various radiolabeled estrogens suggested that multiple estrogens may interact with the receptor. The estrogen-dependent process of sex-reversal of turtle embryos incubated at a male-producing temperature was used to determine whether steroidal estrogens also had synergistic activityin vivo.In this instance, a combination of estriol and estradiol-17β was effective in reversing the gonadal sex of turtle embryos from males to females in a synergistic manner. Our results suggest that the synergy of some combinations of estrogens, synthetic or steroidal, may play a role in the estrogen-dependent process of sexual development in certain species.

Nonparametric regression modelling of green sea turtle growth rates (southern Great Barrier Reef)

Somatic growth rates recorded between 1974 and 1991 for green sea turtles Chelorl~a rnydas resident in the southern Great Barrier Reef (sGBR) foraging grounds of the sGBR genetic stock were modelled using nonparametrlc regression. The sampling deslgn in this long-term mark-recapture program was mixed longitudinal and ~ncluded growth records for female and male turtles 1-anglng between 39 and 116 cm CCL (curved carapace length). Distinct sex-specific growth patterns were found, adults displayed constant negligible growth while inter-annual variability in immature female growth attributable to major oceanographic events was evident. Females grew faster than the males from ca 62 to 65 cm CCL with divergent growth patterns following distinct juvenile growth spurts. The expected female size-specific growth rate function was nonmonotonic, rising from recruitment size (>35 cm CCL) to maximum growth of 2.1 cm yr.' at ca 60 cm CCL before declining to negligible growth approaching sexual maturlty at ca 95 to 100 cm CCL. The male size-specific growth rate function was nonmonotonic rising from the sdme recruitment size to maxlmum groiuth of 2 .2 cm yr.' at ca 63 cm CCL before declining faster than females to negligible grotzlth approachlnq sexual maturity at a significantly smaller size, 595 cm CCL Assumlng a post-hatchling pelagic phase of 5 yr, the mean age at sexual maturity for each sex was estimated at 2-40 yr using numer~cal Integration of the expected slze-specific growth rate functions. Expc.cted age-specific growth rate functions for each sex derlved by numerical differentiat~on showed that the juvenile growth s p u ~ t for females resident in the sGBR foraglng grounds occurs at around 9 yr at large since recruitment (60 cm CCL. 214 yr old) and for males at ca 11 yr a t large (63 cm CCL, 216 yr old). Juvenile growth spurts, sex-specific growth, slow size-specific growth rates, inter-annual growth variability of immatures and many decades to sexual maturity are growth characteristics for green sea turtles resident in the sGBR foraging grounds of the sGBR genetic stock. K E Y WORDS: Somatic growth . Green sea turtles . Great Barrier Reef Nonparametrlc regression

Vitellogenin induction by xenobiotic estrogens in the red-eared turtle and African clawed frog.

Many environmental pollutants have estrogenic activity in animals. Xenobiotic estrogens include many pesticides and industrial chemicals that biocumulate. The impact of these common pollutants on the reproductive success of wildlife may be considerable, particularly in threatened or endangered species. This research examined the use of plasma vitellogenin in males as a biomarker for estrogenic xenobiotics in reptiles and amphibians. Adult male turtles (Trachemys scripta) and frogs (Xenopus laevis) were given ip injections of estradiol-17 beta (E2), diethylstilbestrol (DES), or o,p'-DDT (1-chloro-2-[2,2,2-trichloro-1-(4-chlorophenyl)ethyl)benzene) daily for 7 days, and plasma was collected on day 14. The estrogenic activity of each compound was determined by measuring the induction of plasma vitellogenin. Vitellogenin was identified by precipitation, electrophoresis, Western blot, and enzyme-linked immunosorbant assay (ELISA). In both species, estradiol and DES treatments induced the most vitellogenin, whereas DDT treatments induced smaller amounts of vitellogenin in a dose-dependent fashion. These data indicate that induction of plasma vitellogenin in males may be a useful biomarker of xenobiotic estrogen activity in wild populations of reptiles and amphibians.

Effect of steroid hormones on serotonin, norepinephrine and epinephrine contents in the pineal-paraphyseal complex of the soft-shelled turtle (Lissemys punctata punctata)

Steroids (testosterone, oestrogen, progesterone, corticosterone, dexamethasone and deoxycorticosterone) were administered intramuscularly (0.1 mg.100 g bw-1) on seven consecutive days to juvenile male soft-shelled turtles. Serotonin, norepinephrine and epinephrine contents of the pineal-paraphyseal complex were measured spectrofluorometrically 24 h after the last injection. Testosterone and oestrogen decreased serotonin, norepinephrine and epinephrine levels. Progesterone treatment resulted in an increase of serotonin level and a fall in norepinephrine and epinephrine levels. Corticosterone treatment caused an increase of serotonin level and a decrease of norepinephrine and epinephrine levels. Dexamethasone failed to alter serotonin content, increased norepinephrine and decreased epinephrine levels. Deoxycorticosterone decreased serotonin and elevated epinephrine content.

Male sex steroids and hormonal control of male courtship behavior in the yellow-bellied slider turtle, Trachemys scripta

1. 1. Survey of androgens and estrogens in serum, liver and testes of male yellow-bellied slider turtles, Trachemys (= Pseudemys) scripta, a species exhibiting dissociated gametogenesis age-dependent melanism, revealed the presence of numerous androgen precursors, androgens, androgen metabolites, and estrogens in quantities varying with season, tissue, and male coloration. 2. 2. The most commonly found and abundant androgens in all males were dehydroepiandrosterone (DHEA) and androstenedione, which were present at significantly higher levels in testes than in serum or liver. 3. 3. Epitestosterone was found in the serum and liver of only melanistic males; testosterone was only rarely recovered. 4. 4. Behavioral testing of castrated male turtles implanted with various androgens, as well as intact and sham-operated controls, revealed that melanistic males courted more frequently than nonmelanistic males, and that exogenous testosterone was most effective in inducing courtship behavior in castrated males.

Activation and inhibition of sperm motility by kidney products in the turtle, Trachemys scripta

1. 1. Kidney size in male T. scripta exhibited significant seasonal variation; minimal kidney size was observed during autumn gametogenesis. Histological examination revealed no significant seasonal changes in kidney tubule epithelial height. 2. 2. Renal tubule epithelial cells exhibited pronounced granulation on routine histological examination. Further histochemical tests revealed the granules to test positive for glycogen, acid mucopolysaccharide, protein, lipid and mucin. 3. 3. Some kidney homogenate cytosols were capable of increasing sperm motility when added to suspensions of sperm removed from the ductus deferens of male turtles, but only during the April breeding period. Sperm motility was inhibited in response to kidney cytosols when tested prior to, and following, the spring breeding season. 4. 4. Neither dialysis nor charcoal treatment of activational and inhibitory kidney cytosols affected their ability to activate or inhibit sperm moltility; the inhibitory effects of kidney cytosols were unaffected by heat treatment.

Growth and Morphometrics of the Box Turtle, Terrapene c. carolina

Growth of box turtles (Terrapene c. carolina) in a bottomland forest in Maryland was studied over a period of years (1944-1981). A bivariate analysis of age related growth showed that between 8 and 13 yr, male turtles grew at an average rate of 6.7% per yr in carapace length, whereas females grew at 5.3% per yr. Both males and females grew considerably more slowly between 14 and 19 yr, males at 2.3% per yr and females at 3.4% per yr. Growth slowed still more in the twenties. Growth in the six other dimensions that were measured provided additional comparisons. Allometric analysis of the different carapace and plastron dimensions showed that among males length increased proportionally more than either width or height and that width increased more than height. Among females, only the greater increase of length than of width was statistically significant. Fully grown males were larger than females in all dimensions except height. Differences from the normal scutal pattern occurred in 16.8% of fully grown males and 18.4% of the females.

Pineal 5-methoxytryptophol rhythms in the box turtle: effect of photoperiod and environmental temperature.

The effect of different photoperiods and temperatures on pineal 5-methoxytryptophol (ML) content was investigated in male box turtles, Terrapene carolina triunguis. A rhythm in pineal ML was evident in the long photoperiod (18 h light (L)-6 h dark (D] with high daytime levels of 178 +/- 48 pg/gland (means +/- S.E.M.) which dropped to 38 +/- 6 pg/gland during lights off. In the short photoperiod (8L:16D) no clearcut ML rhythm was observed. Diurnal (10.00-12.00 h) ML concentrations rose linearly (P less than 0.05) with increasing ambient temperatures (5, 15, 20 and 27 degrees C). Day/night differences in ML levels, however, were not significant. Pineal ML in the box turtle thus seems to be modified by the photoperiod and, to a lesser extent, by temperature.

Timing of reproductive behaviour in male musk turtles, Sternotherus odoratus: effects of photoperiod, temperature and testosterone

Although male turtles are usually regarded as having a post-nuptial pattern of gonadogenesis (i.e. gonadal growth dissociated from time of mating), they do mate both in the spring (when testes are regressed, plasma testosterone levels are low, epididymides are enlarged, and temperature and daylength increasing) and in the autumn (testes and epididymides are enlarged, plasma testosterone levels are elevated, and temperature and daylength are decreasing). Therefore different proximate cues may operate in mediating male reproductive behaviour at different times of year. When temperature, photoperiod and testosterone levels were altered in male Sternotherus odoratus during the year in both laboratory and semifield conditions, reproductive behaviour occurred more readily in autumn than in spring (the supposed major breeding period). Short daylength was the key environmental condition stimulating male sexual activity, while temperature changes ‘fine-tuned’ this behaviour. Testosterone was effective in stimulating male behaviour only under certain environmental conditions and males could become refractory to artificially elevated plasma testosterone levels.

Movements and seasonal changes in loggerhead turtle Caretta caretta aggregations in the vicinity of Cape Canaveral, Florida (1978–84)

Abstract Capture records from loggerhead turtles Caretta caretta , tagged in the vicinity of Cape Canaveral, Florida (1978 to 1984), were analysed to determine seasonal distributions and movement patterns. Three distinct groups of turtles (adult males, adult females and subadults) occurred in the study area, and each was dominant at different times of the year. Adult males were most abundant in April and May, adult females were most common from May to July, and subadults constituted over 80% of the population during the remainder of the year. Separate treatment of the three groups was necessary, because movement of one group into the area was apparently correlated with the emigration of the remaining two. The data suggest that nesting females are short-term residents who migrate into the area on two- and three-year intervals and reside elsewhere during non-nesting years. Adult male turtles apparently do not migrate with the females, but may reside in the vicinity of nesting beaches throughout the year. Subadult turtles forage opportunistically along the Atlantic seabord, possibly moving northward as waters warm in the higher latitudes and southward with the onset of winter. Evidence suggests that a resident population of subadults overwinter in the Canaveral area each year.

Effects of environmental contaminants on snapping turtles of a tidal wetland

Snapping turtles (Chelydra serpentina) were collected from a brackish-water and a nearly freshwater area in the contaminated Hackensack Meadowlands of New Jersey and an uncontaminated freshwater area in Maryland to determine the effects of environmental contaminants on a resident wetland species. No turtles were observed or caught in the Meadowlands at two trapping sites that were the most heavily contaminated by metals. Snapping turtles from the brackish-water area had an unusually low lipid content of body fat and reduced growth compared to turtles from the fresh-water areas in New Jersey and Maryland. Despite the serious metal contamination of the Hackensack Meadowlands, the metal content of kidneys and livers from New Jersey turtles was low and not greatly different from that of the Maryland turtles. Organochlorine pesticide concentrations in body fat were generally low at all three study areas. Polychlorinated biphenyls (PCBs) concentrations in fat were highest in male turtles from the New Jersey brackish-water area. Analysis of blood for amino-levulinic acid dehydratase, albumin, glucose, hemoglobin, osmolality, packed cell volume, total protein, triglycerides, and uric acid failed to reveal any differences among groups that would indicate physiological impairment related to contaminants.

Metabolism of testosterone and progesterone by liver homogenates of the seawater turtle (Chelonia mydas mydas) in vitro

The metabolism of testosterone and progesterone was studied in the liver of the sea turtle. Chelonia mydas mydas. Liver homogenate preparations (50 mg/50 ml) from the male turtle were incubated at 25°C for 4 h with either [4- 14C]-testosterone (I) of [4- 14C]-progesterone (II) in a medium supplemented with a NADPH-generating system. Metabolites were isolated and identified by column, thin-layer, and radio gas chromatography. After liquid-liquid partition between petroleum ether-methanol/water, 30% (incubation I) or 7% (incubation II) of the radioactive material was found in the nonpolar phase. In incubation I, the free steroid liberated after treatment with 5% sulphuric acid in methanol was identified as 5β-androstane-3α,17β-diol. The main metabolite of incubation was 5β-androstane-3α,17β-diol. Other metabolites identified were 3β-hydroxy-5β-androstan-17-one, 3α-hydroxy-5β-androstan-17-one, and 5β-androstane-3β,17β-diol. Less than 1% unmetabolized testosterone was recovered. The main metabolite of incubation II was 3α-hydroxy-5β-pregnan-20-one (about 75%). The following steroids were also identifed: 5βprcgnane-3,20-dione, 3β-hydroxy-5β-pregnan-20-one, 5β-pregnane-3α,20β-diol, and 5β-pregnane-3α,20α-diol. Unmetabolized progesterone was less than 1%. The results indicate that under the present conditions the stereospecific ring A reduction of progesterone and testosterone proceeds via a 5β-pathway in liver homogenates of C. mydas. Interestingly, the main metabolite of testosterone is a dihydroxysteroid—5β-androstane-3α,17β-diol—which exists both as the free compound and a fatty acid ester.