We assessed the reproductive ecology of the American crocodile (Crocodylus acutus) on Coiba Island, Panama from January–December 2013. We examined nest site characteristics from January–April and hatchling survivorship from April–December. Ten nests were examined at three nesting localities where 30% of the nests were found under forest canopies and 70% were exposed to sunlight (distance to nearest tree = 280 ± 110 cm). Half of the nests were built closer to the sea and the other half closer to bodies of freshwater (700 ± 360 cm). The nest dimensions were 17.5 ± 7.8 cm from the top of the clutch to the surface, 42.9 ± 9.9 cm from the bottom of the clutch to the surface, and 35.9 ± 3.6 cm wide at the top of the nest cavity. The average soil conditions in the nests consistently had high concentrations of potassium (69.3 mL/L) and manganese (9.2 mg/L), moderate concentrations of phosphorus (6.6 mg/L) and iron (3.7 mg/L), and low concentrations of zinc (0.5 mg/L) and copper (0.0 mL/L). Cation exchange capacity showed consistently high concentrations of calcium (2.2 cmol/kg), moderate of magnesium (1.1 cmol/kg), and low in aluminum (0.1 cmol/L). Volumetric water content was about 25.0 ± 2.6% at the bottom and 22.8 ± .3% in the middle of the clutches. Hatching success was 88.9%, of which 68.3% hatched by themselves or with the mother's aid and 20.6% hatched with our aid. Mean size of the mother was 219 ± 6.2 cm total length (TL) and 115.9 ± 3.0 cm snout-vent length (SVL). The incubation period was estimated to be 85–88 days. TL and SVL growth rate of those individuals were 0.03–0.16 cm/day and 0.00–0.09 cm/day, respectively. Population size was estimated to be 218.6 hatchlings in 22.4 km²; the hatchling population declined 65.7% after the first 2 months (May and June) and 95.9% by July, leaving only 0.5% remaining by December. This is the first study to assess nest-site characteristics and estimate hatchling survival in a Pacific population of American crocodiles.

Although there is much information available about reproduction in Caiman latirostris, knowledge related to steroid hormone levels and follicle development for wild adult females is still lacking. In this study we monitored and assessed the development of follicles and eggs and correlated these results with plasma steroid hormone levels in 32 adult females captured in Santa Fe, Argentina. Fieldwork was carried out over two reproductive seasons (October–January) between 2010 and 2012. Using an ultrasound device to take images of the reproductive structures of adult females, we observed individuals with vitellogenic follicles (n = 5), eggs (n = 4), atretic follicles (n = 11), and inactive reproductive structures (n = 12). We found no reproductive females smaller than 65 cm snout-vent length. High levels of estradiol were found during the ovulation period (November) only in reproductive females. Reproductive females showed no differences in progesterone levels during the study period (November–January) compared to non-reproductive females; however, reproductive females showed higher progesterone levels during nesting (December). We found no differences in progesterone levels between reproductive females and females with atretic follicles at the end of the nesting period (January). Ultrasound imaging was found to be an efficient technique to study reproductive structures at the beginning of reproductive cycle of the broad-snouted caiman. Isolated analyses of hormonal levels are not sufficient to determine the reproductive condition of C. latirostris females.

Male intromittent organs serve two primary reproductive functions: the physical entry into the female body during copulation and the effective delivery of gametes resulting in internal fertilization. Here we present a histological examination of the adult male American alligator phallus semen delivery apparatus, the sulcus spermaticus. While the highly collagenous basal crurae and more distal shaft of the alligator penis contain the rigid structures that facilitate cloacal intromission, the sulcus is more functionally intricate. Here we show the sulcus spermaticus (an open groove that runs along the ventral aspect of the phallic shaft) to be a spatially heterogeneous reproductive structure containing a complex architecture of multiple tissue types. Sulcus morphology markedly changes from its proximal origin between the crurae to its distal exit from the phallus tip. At the proximal origin of the sulcus, the ductus deferens vent semen into an expanded lumen lined by a convoluted secretory epithelium. Along the length of the phallic shaft, an arrangement of longitudinally and radially oriented smooth muscle bundles may act via rhythmic contractions to produce peristaltic sperm conveyance through the sulcus. An extensive vascular network of blood and lymph vessels putatively engorges the sulcus tissues during reproductive activity, increasing tension on an internal network of connective tissues and leading to localized inflation and increased tissue rigidity. We hypothesize that this engorgement works to seal the sulcus groove and allow the structure to convey semen through a functionally closed tube. Further, numerous epithelial secretory cells contribute seminal fluids to the ejaculate and may aid in as yet uncharacterized aspects of sulcus functioning. Together, these observations establish that the sulcus spermaticus is far more than a simple furrow in the phallus shaft for sperm conduction: it contains elements that form a complex functional gamete delivery system.

Studying the mating system of wild populations of American crocodiles, Crocodylus acutus, has important conservation implications. We conducted a preliminary analysis of the mating system of C. acutus in Las Baulas (2007 and 2008), Santa Rosa (2007) and Palo Verde (2008 and 2009) National Parks in Guanacaste, Costa Rica. We captured hatchlings during crocodile surveys and analyzed them with nine polymorphic microsatellite loci to determine relatedness values. High relatedness values indicated that full and half siblings were sampled in a single locality and season. We found full siblings between the years that hatchlings were collected in Las Baulas and Palo Verde National Parks, which suggested mate fidelity. The mate fidelity and high relatedness values could be a consequence of the smaller number of adult crocodiles found within these areas or indicative of a small number of dominant males in the populations. Our results support the need to conduct future studies describing the mating system and nesting success within populations of C. acutus. Understanding of these population factors is crucial to the continued success and maintenance of viable populations of C. acutus.

In some reptiles, gonadal outcome is regulated by temperature during a critical period of the embryonic development. Gonadal steroid hormones are seen as effectors of the gonadal differentiation process. Recently, stress and glucocorticoids (GCs), stress-related hormones in vertebrates, have been considered as potential modulators of the sex determination process in some vertebrates that present temperature-dependent sex determination (TSD). In reptiles, corticosterone is the main GC produced, and its administration to eggs causes a bias in sex ratio in some lizards. In this context, we aim at assessing whether dexamethasone (Dex), a potent synthetic glucocorticoid, can modify the sex ratio in Caiman latirostris, a species with strong TSD. As a first step, we incubated embryos at masculinizing temperatures (33°C; 100% males). Different doses of Dex were topically applied to the eggshell at stage 20, prior to gonadal differentiation. We assessed embryonic development at stages 22 and 25 and evaluated some physiological and morphological hatchling traits. Embryonic mortality was not affected by dexamethasone manipulation. No effects of Dex on sex ratio were found and all animals analyzed histologically possessed testes. However, older embryos and hatchlings from Dex treated eggs were heavier, larger, and hatched earlier than control individuals. Our results do not account for Dex involvement in the process of ovarian differentiation, at least under a strong masculinizing temperature. Nevertheless, they suggest that Dex might accelerate embryo development by enhancing intermediate metabolism and/or by stimulating growth hormone secretion.

The temperature-sensitive period is the time during development during which sex determination occurs in vertebrates that undergo temperature-dependent sex determination, such as in caimans. The interplay among temperature and steroid hormones is also known, and it has been suggested that stress hormones (corticosterone) might influence sex ratios in some reptiles. To explore this, we measured the levels of corticosterone in Caiman latirostris to determine if incubation temperature (31°C, 33°C, and 34°C) affects plasma corticosterone levels. Differences among nests were observed in plasma corticosterone. However, hormone levels showed no significant differences between sexes or incubation temperatures in Caiman latirostris embryos or hatchlings. Corticosterone levels were 0.01–2.2 ng/ mL in embryos incubated at 31°C (100% of females), 0.01–4.65 ng/mL in those incubated at 33°C (100% of males), and 0.01–6.31 ng/mL in embryos incubated at 34°C (100% of males). Corticosterone levels were higher in hatchlings, being 1.11–39.18 in those produced at 31°C, 2.85–22.36 at 33°C, and 2.72–39.05 ng/mL at 34°C.

Very little is known about the attainment of puberty in reptiles. In the American alligator (Alligator mississippiensis) males are assumed to be sexually mature at about 1.8 m in total length, but it is not clear at what size they produce testosterone, spermatozoa and mate successfully. We re-examined this question by studying plasma testosterone levels in blood samples from a large sample of alligators (∼1,500) collected every month of the year and ranging in size from approximately 61 cm (2 ft) to 360 cm (11.5 ft). Testosterone values ranged from 0.05–115.41 ng/mL. All size classes of alligators exhibited a seasonal cycle in testosterone levels, but the concentrations were size-dependent: the larger the alligator the higher the testosterone. In all size-classes testosterone reached a peak in the breeding season (March–May). Mean testosterone in the largest size-class during breeding was 75 ng/mL whereas in the smallest size-class peak testosterone was less than 3 ng/mL. The smallest size-class (59–89 cm) showed an additional rise in testosterone in late summer. The attainment of sexual maturity in alligators appears to be closely associated with growth and is a gradual process lasting several years. Sexually immature alligators show a seasonal pattern of testosterone secretion similar to that of adults, but the values are significantly lower.