How do maternal androgens and estrogens affect sex determination in reptiles with temperature-dependent sex?

Temperature sex determination (TSD) in reptiles has been studied to elucidate the mechanisms by which temperature is transformed into a biological signal that determines the sex of the embryo. Temperature is thought to trigger signals that alter gene expression and hormone metabolism, which will determine the development of female or male gonads. In this review, we focus on collecting and discussing important and recent information on the role of maternal steroid hormones in sex determination in oviparous reptiles such as crocodiles, turtles, and lizards that possess TSD. In particular, we focus on maternal androgens and estrogens deposited in the egg yolk and their metabolites that could also influence the sex of offspring. Finally, we suggest guidelines for future research to help clarify the link between maternal steroid hormones and offspring sex.

Caiman's fat enriched with n-3 fatty acids: potential food supplement.

Broad-snouted caiman (Caiman latirostris) products (meat, fat and oil) are currently beginning to be valued as a food of special interest due to its high content of n-3 fatty acids. Thus, the objective of this study was to characterize the fats of caiman fed with diets enriched with flaxseeds (Linus usitatissimun) rich in n-3 fatty acids, lignans and antioxidants. Caimans were fed six days a week with: a control diet (C), and a diet enriched with ground flaxseed = 90% C + 10% flaxseed ground (FS), during 30 (FS30) and 60 (FS60) days. Animals fed the flaxseed-enriched diets increased linolenic acid content and reduced the n-6/n-3 ratio of fats relative to controls, and this improvement increased over time. The proportion of eicosapentaenoic acid also increased, but there was no difference at the time the enriched diets were offered. Caiman fat of the FS30 and FS60, showed a decrease in lipoperoxidation (24% and 40%) and reactive oxygen species (44% and 76%) accompanied by an increase in antioxidant systems. Consumption of a flax-enriched diet by caimans increases the content of essential fatty acids and improves the lipoperoxidative status of fat. This provides an enriched fat with potential for the development products for human consumption.

Body Condition of Reproductive and Non-Reproductive Broad-Snouted Caiman Females.

In this work, we calculated the body condition indices, K-Fulton and scaled mass index (SMI), of reproductive and non-reproductive Caiman latirostris adult females as an indication of stored energy. We considered 87 adult females captured from 2001 to 2018, both reproductive and non-reproductive. The body condition was calculated considering two scenarios: (a) only the weight of the female, and (b) the sum of the weight of the female and the average dry weight of her nest. We tested the difference in body condition between reproductive and non-reproductive females. We also evaluated the minimal body condition required to guarantee that females above it are reproductive by drawing a line that separated the body condition of strictly reproductive individuals from those that may or may not be reproductive. Reproductive females had better body condition than non-reproductive ones. Our SMI.S line separated almost 70% of the reproductive females. Based on our results, we can guarantee that a female whose body condition is above the line will reproduce, although not all those females below the line are non-reproductive, as a few of those under the line will nest. With this information, we have one more biological indicator to take into account when making management and conservation decisions.

Preceding anti-spike IgG levels predicted risk and severity of COVID-19 during the Omicron-dominant wave in Santa Fe city, Argentina.

The SARS-CoV-2 Omicron variant has increased infectivity and immune escape compared with previous variants, and caused the surge of massive COVID-19 waves globally. Despite a vast majority (~90%) of the population of Santa Fe city, Argentina had been vaccinated and/or had been infected by SARS-CoV-2 when Omicron emerged, the epidemic wave that followed its arrival was by far the largest one experienced in the city. A serosurvey conducted prior to the arrival of Omicron allowed to assess the acquired humoral defences preceding the wave and to conduct a longitudinal study to provide individual-level real-world data linking antibody levels and protection against COVID-19 during the wave. A very large proportion of 1455 sampled individuals had immunological memory against COVID-19 at the arrival of Omicron (almost 90%), and about half (48.9%) had high anti-spike immunoglobulin G levels (>200 UI/ml). However, the antibody titres varied greatly among the participants, and such variability depended mainly on the vaccine platform received, on having had COVID-19 previously and on the number of days elapsed since last antigen exposure (vaccine shot or natural infection). A follow-up of 514 participants provided real-world evidence of antibody-mediated protection against COVID-19 during a period of high risk of exposure to an immune-escaping highly transmissible variant. Pre-wave antibody titres were strongly negatively associated with COVID-19 incidence and severity of symptoms during the wave. Also, receiving a vaccine shot during the follow-up period reduced the COVID-19 risk drastically (15-fold). These results highlight the importance of maintaining high defences through vaccination at times of high risk of exposure to immune-escaping variants.

Genital anatomy and copulatory interactions in the broad snouted Caiman (Caiman latirostris).

The broad snouted caiman is a crocodylian native to South America that is subject to extensive conservation management in both wild and farming environments. Although reproductive behaviors like egg laying and clutch care have been examined in this species, little else is known about their copulatory system. We examined the anatomy of male and female cloacal and genital tissues ex vivo to build hypotheses of their interactions during copulation and the effects of that interaction on insemination. Male phallic glans tissues were artificially inflated to expand into their copulatory state, allowing the examination and quantification of structural changes at the gross and tissue levels. Digital reconstruction of MRI stacks yielded three-dimensional tissue compartment specific glans models of the inflated state. Silicone molds of female cloacae and oviducts in conjunction with dissection and diceCT analysis allowed us to assess internal geometry and infer how male and female features interact in copulo. We observed glans expansion within the female proctodeum would result in a copulatory lock limiting deeper intromission or retraction. Intromission and subsequent creation of the copulatory lock produces extensive clitoral compression, providing a possible mechanism for female assessment of male copulatory performance. Further, glans expansion forms a distal lumen that positions the glans tip in or near the vaginal openings. A coiled, muscular vagina provides a possible mechanism for postcopulatory sexual selection by excluding semen. Together, the complex male-female interaction supports evidence for cryptic selection by female choice, which can act as a driver of genital coevolution.

Effect of flaxseed and flaxseed oil supplemented in caiman diet on meat fatty acids.

Increasing polyunsaturated or long-chain fatty acids in meat for human consumption improves both nutritional quality and consumer perception. The increase could occur through the addition of rich sources of omega-3 fatty acids (such as flaxseed or flaxseed oil) to the animal diet. The aim of this study was to evaluate the effects of dietary supplement with two presentations of flax (crushed seeds or oil) on the change of FA content in two cuts of caiman meat (tail and neck). We measured fatty profile in two different caiman meat cuts (neck and tail) from 30 animals (total length 96.7 ± 4.9 cm, snout-vent length 47.8 ± 3 cm, weight 4.2 ± 0.6 kg), raised in individual enclosures, fed three a week for 50 days with crushed chicken head and a dry food formulated for these reptiles in a 70/30 ratio (C, n = 10), control diet with 10% crushed flaxseed (FS, n = 10), and control diet with 10% flaxseed oil (FO, n = 10), while the remaining days animals were fed the control diet. Meats from animals fed both enrichment diet (FS and FO) showed an increase of C18:3n-3 and ΣUFA with respect to control diet. Although both enriched diets raised the levels of C18:3n-3, the neck showed higher values than the tail. We observed that the neck is more susceptible than the tail to be improved by FO, which could suggest that it is more beneficial to consume neck meat. In order to be implemented in caiman farms, flaxseed oil is more expensive than seed, but more effective, easier to manage, and is practical for application on caiman farms.

Optimization of oil extraction from caiman fat. Characterization for use as food supplement.

The leather of Caiman latirostris is highly appreciated in the fashion industry and the meat is valued as an important food but its fat are usually discarded because it has no commercial value. However it is an alternative source of essential fatty acids and could be used for human consumption. The aim was to optimize the oil extraction from Caiman latirostris fat and to carry out the chemical and microbiological characterization for its use as food supplement. The oil obtained by fusion method contains fatty acids with high nutritional quality such as oleic acid (34%), linoleic acid (30%) and α-linolenic acid (2%). The atherogenicity index was 0.29 and the thrombogenicity index 0.47. The presence of mesophilic aerobic bacteria, coliform bacteria, Escherichia coli and Salmonella were not observed, and the oil is stable for 4 months at 25 °C and for at least 8 months in an inert atmosphere at 25 °C.

"Intracascaral space" an eggshell structure of Caiman latirostris eggs.

In recent decades, eggshells of eggs from large-bodied reptiles have been studied by many researchers, to describe the eggshell, to compare them to extinct lineages that once inhabited our planet and also to understand how the egg provides the embryo specific conditions during incubation. In previous studies we described and characterized normal and pathologic Caiman latirostris eggshells; we also evaluated how the eggshell changes during incubation. In a study relating temperature variation and eggshell structures of successful eggs, we observed empty structures not previously described that we termed "intracascaral space". The aim of this study is to describe this structure of C. latirostris eggshells. We hypothesize about the possible functions which it would perform during incubation and for development of the embryos.

Presence of Leptospira spp. in Caiman latirostris (Crocodylia, Alligatoridae) populations in Santa Fe, Argentina.

Leptospirosis is a disease caused by pathogenic spirochetes of the genus Leptospira, transmitted by wild and domestic animals. Rodents play a fundamental role in the transmission cycle of this zoonosis but the function of reptiles is unknown. For example, crocodilians could play an important role in the transmission of this disease by living in ideal environments (bodies of shallow water and high temperatures) for the colonization of this bacterium. However, few studies have documented the presence of zoonotic diseases in caiman populations. Our objective was to assess the prevalence of antibodies to leptospira and the presence of Leptospira spp. in wild and captive Caiman latirostris. Blood samples were taken from 45 individuals (20 wild and 25 captive). Before extraction, we cleaned each caiman's neck in order to prevent contamination of samples. We determined the presence of antibodies in serum by microscopic agglutination test (MAT) and polymerase chain reaction (PCR) to detect DNA of the bacteria. We excluded 9 of the 45 samples analyzed by MAT because 5 had lipemic serum and 4 were contaminated (colonized by other organisms). Of the 36 caimans studied by microscopic agglutination test (MAT), 56% (20/36) were considered reactive (titers ≥50). In 74% (14/19) of captive samples and 35% (6/17) of wild samples, antibodies to leptospira were detected by MAT. The serogroup with highest occurrence was Pyrogenes (85%, n = 17/20), presenting coagglutinations with Icterohaemorrhagiae (25%, n = 5/20). One sample from a captive animal was positive for PCR, and we could not isolate leptospires because of agar contamination. Of the 45 blood agar media, 17.8% were contaminated and the rest were negative. This work determined the presence of Leptospira spp. in one caiman and a high prevalence of antibodies in captive caiman relative to wild individuals.

Influence of temperature variation on incubation period, hatching success, sex ratio, and phenotypes in Caiman latirostris.

Temperature is crucial for reptiles, also during embryonic development, particularly for species with temperature-dependent sex determination. Under natural conditions, Broad-snouted caiman (Caiman latirostris) eggs are influenced by thermal changes in the interior of the nest related to the external environmental temperature. As nests are subject to variations in temperature and most lab studies on crocodilian incubation have been carried out at constant temperatures, we were interested in determining how temperature fluctuations may affect the development of caiman embryos. We investigated the effects of incubation at constant temperatures (31°C, 32°C, and 33°C) and fluctuating temperatures (31 ± 2, 32 ± 1, and 32 ± 2°C) on the following aspects: incubation period duration, hatching success, sex ratio, total length, and body mass of C. latirostris hatchlings. Eggs incubated at 31°C produced 100% females, those at 32°C produced 71.6% females (however, the sex ratio was nest related), and at 33°C produced 100% males. We found a masculinizing effect when incubation was at 31 ± 2°C compared with a constant 31°C; and temperature fluctuations at 32°C (32 ± 1 and 32 ± 2°C) had a negative effect on hatchlings size and mass, and hatching success compared with constant incubation temperatures of 32°C and 33°C. Finally, the effect of temperature variation during the incubation period on sex ratio, hatching success, and phenotype depends on the mean temperature, as the fluctuation around 31°C affected the sex ratios and incubation period, and the fluctuation around 32°C affected hatchling success and size.

Embryological development of Salvator merianae (Squamata: Teiidae).

Here we describe the embryonic development of Salvator merianae external morphologic features, as based on observation of 28 embryos across different days of incubation at 31 ± 0.5°C. Observed developmental stages were grouped and classified into the early, middle, and late periods. The early period (Stages 3-11) is distinguished by the origin of the encephalic vesicles, sensory placodes, pharyngeal arches, and degree of body flexion and rotation. The medium period (Stages 8-15) is distinguished by limb differentiation and by cranium-facial characteristics. The late period (Stages 15-18) is determined by scale patterns, pigmentation, and embryo growth.

Eggshell structure in Caiman latirostris eggs improves embryo survival during nest inundation.

Egg inundation often results in poor hatching success in crocodylians. However, how tolerant eggs are to submergence, and/or how eggshell ultrastructure may affect embryo survival when inundated, are not well understood. In this study, our objective was to determine if embryo survival in Caiman latirostris is affected by eggshell surface roughness, when eggs are submerged under water. Tolerance to inundation was tested early (day 30) versus late (day 60) in development, using eight clutches (four per time treatments), subdivided into four groups: (N = 9 per clutch per treatment; 9 × 4 = 36 eggs per group). 'Rough' eggshell represented the natural, unmodified eggshell surface structure. 'Smooth' eggshell surface structure was created by mechanically sanding the natural rough surface to remove surface columnar elements and secondary layer features, e.g. irregularities that result in 'roughness'. When inundated by submerging eggs under water for 10 h at day 30, 'smooth' eggshell structure resulted in more than twice as many dead embryos (16 versus 6, smooth versus rough; N = 36), and fewer than half as many healthy embryos (6 versus 13, smooth versus rough, respectively; N = 36). By contrast, at day 60, inundation resulted in very low hatching success, regardless of eggshell surface structure. Only two hatchlings survived the inundation, notably in the untreated group with intact, rough eggshells. Inundation produced a high rate of malformations (58% at day 30), but did not affect hatchling size. Our results indicate that eggshell roughness enhances embryo survival when eggs are inundated early in development, but not late in development. Apparently, the natural surface 'roughness' entraps air bubbles at the eggshell surface during inundation, thereby facilitating gas exchange through the eggshell even when the egg is submerged under water.

Irregularly calcified eggs and eggshells of Caiman latirostris (Alligatoridae: Crocodylia).

We describe irregularly calcified egg and eggshell morphologies for the first time in nests of the broad-snouted caiman, Caiman latirostris. Research is based on detailed descriptions of 270 eggs from a total sample of 46,800 collected between 2005 and 2011 in Santa Fe Province, Argentina, and encompasses animals from both natural habitats and held in captivity. We discuss possible reasons for the occurrence of eggs with different mineralisation patterns in our extensive C. latirostris field sample and its conservation significance; the chemistry of egg laying in amniotes is sensitive to environmental contamination which, in turn, has biological implications. Based on our egg sample, we identify two caiman eggshell abnormalities: (1) regularly calcified eggs with either calcitic nodules or superficial wrinkles at one egg end and (2) irregularly calcified eggs with structural gaps that weaken the shell. Some recently laid clutches we examined included eggs with most of the shell broken and detached from the flexible membrane. Most type 1 regularly calcified eggs lost their initial calcified nodules during incubation, suggesting that these deposits do not affect embryo survival rates. In contrast, irregularly calcified caiman eggs have a mean hatching success rate of 8.9% (range 0-38%) across our sample compared to a mean normal success of 75%. Most irregularly calcified caiman eggs probably die because of infections caused by fungi and bacteria in the organic nest material, although another possible explanation that merits further investigation could be an increase in permeability, leading to embryo dehydration.