Ultrasonographic and radiographic evaluation of gestation in golden lanceheads (Bothrops insularis) in ex situ breeding programs.

The golden lancehead (Bothrops insularis) is a critically endangered snake endemic to Queimada Grande Island, southeastern Brazil. Captive breeding programs are vital for the conservation of this species. This study evaluates the gestation of two females golden lanceheads using radiography and ultrasonography. The first female was collected on the island while reproductively active (late vitellogenesis or early pregnancy) and kept in captivity. This female gave birth to five neonates after an 8-month gestation period, which is longer than estimates based on specimens preserved in museums. The second female copulated in captivity in July and probably ovulated in October. In this case, no embryonic development was detected, and the female deposited 14 undeveloped eggs approximately 6 months after ovulation.

Reproductive cycles of neotropical boid snakes evaluated by ultrasound.

Snakes have increasingly been bred as pets around the world. Few studies have addressed the reproduction of boid snakes, and no study has addressed their reproductive cycles in captivity. Thus, this paper describes the reproductive aspects of Brazilian boids in captivity. We used ultrasonography to characterize the reproductive cycle of four boid species in captivity in the Southern Hemisphere: the anaconda (Eunectes murinus), the red-tailed boa (Boa constrictor constrictor), the Amazon tree boa (Corallus hortulanus), and the rainbow boa (Epicrates cenchria). Nonvitellogenic follicles occurred from January to December in anaconda and red-tailed boa and for a shorter period from September to February in Amazon tree boa and from January to May in rainbow boa. Vitellogenesis occurred from late June to late March in E. murinus in year-round (12 months), from March to March in Amazon tree boa, from late September to late March in red-tailed boa, and from late March to late September in rainbow boa. Mating occurred from late March to late September in red-tailed boa and rainbow boa and from late September to late March in Amazon tree boa. No mating was observed in anacondas, but a female probably underwent parthenogenesis. Births occurred in July in anaconda and in March to July in Amazon tree boa and from December to March in red-tailed boa and rainbow boa. In males, increases in testicular size were associated with the mating season. Ultrasonography proved to be a safe and noninvasive technique to study the reproductive cycle of giant snakes in captivity.

Short-term cold storage of seminal samples of the golden lancehead pitviper (Bothrops insularis).

Assisted reproductive technologies such as cooling, freezing, and artificial insemination are essential in conserving genetic diversity of critically endangered reptiles like the golden lancehead pitviper (Bothrops insularis). Therefore, we examined viability of semen samples from captive golden lanceheads diluted in coconut-water based extender over a 48-h period of cooling (5°C). Semen evaluations were performed immediately after the dilution and at 6, 24, and 48 h, using computer-assisted sperm analysis and stains to assess plasma membrane and acrosomal status. Our findings showed that the extender and protocol employed here were effective in preserving golden lancehead pitviper spermatozoa for a short periods of time (48 h), allowing semen samples to be transported to distant locations for immediate use without the setbacks of cryopreservation.

Reproductive cycles of two island pitvipers species (Serpentes, Viperidae) determined by ultrasonography and radiography.

We used ultrasonography and radiography to assess the sexual organs and characterize the reproductive cycle of captive golden lancehead (Bothrops insularis) and Alcatrazes lancehead (B. alcatraz), two endangered island snake species in Brazil. We assessed 46- individuals of golden lancehead and 12 of Alcatrazes lancehead kept in captivity between 2014 and 2020. Follicular development was similar between species, but follicles in Alcatrazes lancehead were smaller than in the golden lanceheads. Female golden lanceheads produced 24 live young, seven stillborn and 73 undeveloped eggs. Parturition of live young occurred between midsummer (February) and early autumn and gestation averaged 8 months. Female Alcatrazes lanceheads produced four live young in midsummer, and one undeveloped egg in early autumn. Males and females of both species have seasonal and biennial reproductive cycles. Sperm storage in both sexes is essential to coordinate male and female cycles. The data obtained with golden lancehead and Alcatrazes lancehead in captivity, demonstrate a degree of conservatism, following data from other Bothrops.

Male genital system of Ameiva ameiva (Squamata: Teiidae).

Understanding squamate reproductive morphology is crucial for investigating ecological, behavioral, and evolutionary questions. Here, we describe the anatomy and histology of the male genital system of Ameiva ameiva from southeastern Brazil. Ten adult males were dissected to characterize genital macroscopy and collect fragments of the testes, gonadoducts, and kidneys for histological examination. We examined 10 transverse histological sections per individual and measured the epithelial height of the epididymis and ductus deferens. The male reproductive system consists of a pair of yellowish oval testes, the rete testis, ductuli efferentes, epididymis, ductus deferens, ampulla ductus deferentis, sexual segment of the kidney (SSK), cloaca, and hemipenis. The hemipenis is elongated, cylindrical, and unilobed, with a sulcate face and an asulcate face, which has continuous fringes throughout its length. Seminiferous tubules exhibited germ cells at various stages. The epididymis is wider and more coiled than the ductus deferens. The rete testis has a simple squamous epithelium with long stereocilia, while the narrower ductuli efferentes are lined by a simple ciliated cuboidal epithelium. The epididymal epithelium is pseudostratified columnar, with basal and ciliated principal cells, whereas the ductus deferens epithelium is pseudostratified to simple cuboidal. The epididymal epithelium is 1.5 times taller than the ductus deferens epithelium. Here, we observed the SSK present in the cortex of the ventral region of the kidneys due to the hypertrophy of the distal convoluted tubules, as well as its secretory activity. Our findings will contribute to future research into the evolution of squamate reproductive morphology.

Study of defensive behavior of a venomous snake as a new approach to understand snakebite.

Snakebites affect millions of people worldwide. The majority of research and management about snakebites focus on venom and antivenom, with less attention given to snake ecology. The fundamental factor in snakebites is the snakes' defensive biting behavior. Herein we examine the effects of environmental variables (temperature, time of day, and human stimulus) and biological variables (sex and body size) on the biting behavior of a medically significant pit viper species in Brazil, Bothrops jararaca (Viperidae), and associate it with the epidemiology of snakebites. Through experimental simulations of encounters between humans and snakes, we obtained behavioral models applicable to epidemiological situations in the State of São Paulo, Brazil. We found a significant overlap between behavioral, morphological, environmental, and epidemiological data. Variables that increase snakebites in epidemiological data also enhance the tendency of snakes to bite defensively, resulting in snakebites. We propose that snakebite incidents are influenced by environmental and morphological factors, affecting the behavior of snakes and the proportion of incidents. Thus, investigating behavior of snakes related to snakebite incidents is a valuable tool for a better understanding of the epidemiology of these events, helping the prediction and, thus, prevention of snakebites.

Reproductive maturity and sexual dimorphism of a population of Amerotyphlops brongersmianus from a Restinga area in southeastern Brazil (Serpentes: Typhlopidae).

Natural history data are important for a better understanding of distinct aspects of snake biology, and this information in scarce on Scolecophidia. Here we focus on sexual maturity and sexual dimorphism within a population of Amerotyphlops brongersmianus from the Restinga de Jurubatiba National Park, Rio de Janeiro state, Brazil. The smallest sexually active male and female showed snout-vent length of 117.5 and 158.4 mm, respectively. Females had statistically significant larger body and head length sizes, whereas males had longer tails. Juveniles showed no sexual dimorphism for any analyzed feature. Secondary vitellogenic follicles had a more opaque, yellowish/darker aspect, being larger than 3.5 mm. We reinforce that in addition to traditional features for determining sexual maturity, morphological and histological characteristics of kidneys should be evaluated in males, as well as the morphology of the infundibulum in females. Histological data show development of seminiferous tubules and presence of spermatozoa in males, and infundibulum receptacles and uterine glands in females as a sign of sexual maturity. This type of information is essential for a more accurate description of data on sexual maturity, allowing access to information on the development of reproductive structures that are not available macroscopically.

Morphological and structural differences between the hemipenes and hemiclitores of golden lancehead snakes, Bothrops insularis (Amaral, 1922), revealed by radiography.

The golden lancehead (Bothrops insularis) is a critically endangered and island endemic snake. A remarkable characteristic of golden lanceheads is the high frequency of females showing hemiclitores. Here, we use radiography to identify morphological and structural differences between the hemipenes and hemiclitores in captive individuals. Sex was identified using the probing method and confirmed by visualizing the gonads by ultrasound. Hemipenes appeared as herringbone-shaped structures with bone radiopacity and hemiclitores as triangular structures with soft tissue radiopacity. These differences suggest that hemiclitores spines fail to develop the level of ossification observed in hemipenis spines.

Morphology and seasonality of the sexual segment of the kidney in genus Bothrops snakes.

In Squamata, the sexual segment of the male kidney (SSK) produces secretory granules that are transmitted to the female tract during copulation. Here, we investigate the morphology of SSK in various species of the pitviper-genus Bothrops, a medically relevant clade of Neotropical snakes, using histology, histochemistry, as well as scanning and transmission electron microscopy. We also evaluated the relationship of SSK hypertrophy with spermatogenesis and storage of sperm. The SSK in Bothrops consists of tall columnar epithelial cells filled with electron-dense secretory granules. These granules are apparently released into the lumen by an apocrine secretory process. In general, the ultrastructural morphology of SSK in Bothrops is similar to those described for other pitvipers in the genus Agkistrodon. In most Bothrops males, the SSK produces carbohydrates and proteins, and occurs in all lobes of the kidney, occupying most of the kidney mass. In contrast, intersexual females (with male copulatory organs) of Bothrops insularis had a smaller SSK diameter than males and did not show hypertrophy. The seasonal development of SSK in Bothrops species seems to be driven by environmental factors rather than phylogeny, since sister species exhibited periods of hypertrophy at different months. In most Bothrops males, hypertrophy occurs at various seasons of the year (spring, summer, autumn), even in winter, and seasonal changes in SSK do not necessarily accompany spermatogenesis.

Evidence of facultative parthenogenesis in three Neotropical pitviper species of the Bothrops atrox group.

We examined four suspected cases of facultative parthenogenesis in three species of a neotropical lineage of pitvipers of the Bothrops atrox group. Reproduction without mating was observed in captive females of B. atrox, B. moojeni and B. leucurus housed alone for seven years (the two former species) and nine years (the latter one). In addition to the observation of captivity data, we investigated molecularly this phenomenon using heterologous microsatellites. DNA was extracted from the mothers' scales or liver, from embryo and newborn fragments, and yolked ova. Four of the microsatellites showed good amplification using Polymerase Chain Reaction and informative band segregation patterns among each mother and respective offspring. Captivity information, litter characteristics (comparison of the number of newborns, embryos and yolked ova) and molecular data altogether agreed with facultative parthenogenesis predictions in at least three out of the four mothers studied: B. atrox (ID#933) was heterozygous for three out of the four markers, and the sons S1 and S2 were homozygous; B. moojeni (BUT86) was heterozygous for two out of four markers, offspring S1, S3, E2, and E4, and O1 to O6 were homozygous; and B. leucurus (MJJS503) was heterozygous for three out of four markers, and son E1 and O1 were homozygous. B. moojeni (BUT44) was homozygous for all loci analyzed in the mother and offspring, which although not informative is also consistent with parthenogenesis. This study represents the first molecular confirmation of different pitviper species undergoing facultative parthenogenesis among Neotropical endemic snakes.

Reproductive cycles of two island pitvipers species (Serpentes, Viperidae) determined by ultrasonography and radiography

We used ultrasonography and radiography to assess the sexual organs and char-acterize the reproductive cycle of captive golden lancehead (Bothrops insularis) and Alcatrazes lancehead (B. alcatraz), two endangered island snake species in Brazil. We assessed 46- individuals of golden lancehead and 12 of Alcatrazes lancehead kept in captivity between 2014 and 2020. Follicular development was similar between species, but follicles in Alcatrazes lancehead were smaller than in the golden lance-heads. Female golden lanceheads produced 24 live young, seven stillborn and 73 un-developed eggs. Parturition of live young occurred between midsummer (February) and early autumn and gestation averaged 8 months. Female Alcatrazes lanceheads produced four live young in midsummer, and one undeveloped egg in early autumn. Males and females of both species have seasonal and biennial reproductive cycles. Sperm storage in both sexes is essential to coordinate male and female cycles. The data obtained with golden lancehead and Alcatrazes lancehead in captivity, demon-strate a degree of conservatism, following data from other Bothrops.

Male genital system of Ameiva ameiva (Squamata: Teiidae)

Understanding squamate reproductive morphology is crucial for investigating ecological, behavioral, and evolutionary questions. Here, we describe the anatomy and histology of the male genital system of Ameiva ameiva from southeastern Brazil. Ten adult males were dissected to characterize genital macroscopy and collect fragments of the testes, gonadoducts, and kidneys for histological examination. We examined 10 transverse histological sections per individual and measured the epithelial height of the epididymis and ductus deferens. The male reproductive system consists of a pair of yellowish oval testes, the rete testis, ductuli efferentes, epididymis, ductus deferens, ampulla ductus deferentis, sexual segment of the kidney (SSK), cloaca, and hemipenis. The hemipenis is elongated, cylindrical, and unilobed, with a sulcate face and an asulcate face, which has continuous fringes throughout its length. Seminiferous tubules exhibited germ cells at various stages. The epididymis is wider and more coiled than the ductus deferens. The rete testis has a simple squamous epithelium with long stereocilia, while the narrower ductuli efferentes are lined by a simple ciliated cuboidal epithelium. The epididymal epithelium is pseudostratified columnar, with basal and ciliated principal cells, whereas the ductus deferens epithelium is pseudostratified to simple cuboidal. The epididymal epithelium is 1.5 times taller than the ductus deferens epithelium. Here, we observed the SSK present in the cortex of the ventral region of the kidneys due to the hypertrophy of the distal convoluted tubules, as well as its secretory activity. Our findings will contribute to future research into the evolution of squamate reproductive morphology.

Cycle of the sexual segment of the kidney: histological insights into the role of the urinary tract in the reproduction of male Notomabuya frenata (Squamata: Scincidae)

The kidneys of male Squamata have an important reproductive function as some portions of the nephron may undergo hypertrophy, characterizing the sexual segment of the kidney (SSK). Although its function is still not completely understood, it is believed that the secretions produced by the SSK may act in the maintenance of spermatozoa. In this study, we investigated the reproductive biology of males of Notomabuya frenata based on the seasonal variation of the SSK. We performed macroscopic and microscopic evaluations of the male reproductive tract of museum specimens to characterize the SSK cycle. The nephron portion in which hypertrophy was observed was the collecting duct with secretory granules accumulation in the apical portion. SSK hypertrophy was observed in all seasons, with the tubule diameter in autumn differing from spring and the epithelium height showing no variation. Alcian Blue reacted positively to acid mucopolysaccharides in all seasons. Periodic acid–Schiff’s reacted positively to neutral mucopolysaccharides in all seasons, except autumn. Both stains reacted only in the collecting duct. In addition, spermatozoa were found in the lumen of the SSK of one specimen examined. Cycle of the SSK varied seasonally as does the chemical composition of the secretions produced by the collecting duct. The reflux of spermatozoa into SSK may indicate that (1) these secretions act in sperm maintenance, and (2) possibly there is communication between the seminal and urinary ducts.

Study of defensive behavior of a venomous snake as a new approach to understand snakebite

Snakebites affect millions of people worldwide. The majority of research and management about snakebites focus on venom and antivenom, with less attention given to snake ecology. The fundamental factor in snakebites is the snakes’ defensive biting behavior. Herein we examine the effects of environmental variables (temperature, time of day, and human stimulus) and biological variables (sex and body size) on the biting behavior of a medically significant pit viper species in Brazil, Bothrops jararaca (Viperidae), and associate it with the epidemiology of snakebites. Through experimental simulations of encounters between humans and snakes, we obtained behavioral models applicable to epidemiological situations in the State of São Paulo, Brazil. We found a significant overlap between behavioral, morphological, environmental, and epidemiological data. Variables that increase snakebites in epidemiological data also enhance the tendency of snakes to bite defensively, resulting in snakebites. We propose that snakebite incidents are influenced by environmental and morphological factors, affecting the behavior of snakes and the proportion of incidents. Thus, investigating behavior of snakes related to snakebite incidents is a valuable tool for a better understanding of the epidemiology of these events, helping the prediction and, thus, prevention of snakebites.

Sperm storage in Crotalus durissus (Serpentes: Crotalinae): histological insights about the female reproductive tract of pit vipers

The reproductive cycle of Crotalus durissus is markedly seasonal and synchronous between individuals. The start of vitellogenesis occurs at the end of the summer and coincides with copulation. However, given that the copulation is dissociated from ovulation, sperm storage is obligatory in females. In viperids, sperm storage in the female reproductive tract is reported to occur in two regions: (1) the posterior infundibulum, which presents sperm storage glands; and (2) the nonglandular uterus where sperm is stored in crypts by means of the uterine muscular twisting (UMT). The mechanisms that allow the survival of sperm in the female reproductive tract of snakes are still unknown. In this study, we investigated five regions of the reproductive tract of C. durissus, searching for the presence of spermatozoa and sperm storage structures in different oviductal portions. Additionally, we used histological techniques to verify the occurrence of hypertrophy of the infundibular and uterine glands during the processes of vitellogenesis, as well as histochemical techniques to investigate the nature of the secretion produced in the nonglandular uterus and posterior infundibulum. Storage sperm were observed in the nonglandular uterus and although the posterior infundibulum had storage receptacles, sperm were not observed in that region. Both sperm storage regions presented granules testing positive for acidic and neutral polysaccharides, in vitellogenic and previtellogenic females. This presence of guaranteeing conditions for sperm storage. Histochemical analysis revealed the possible storage capacity of sperm in the nonglandular uterus. In addition, the UMT was observed in all the females with storage sperm, which assures the maintenance of sperm in the nonglandular uterus until ovulation.

Reproductive maturity and sexual dimorphism of a population of Amerotyphlops brongersmianus from a Restinga area in southeastern Brazil (Serpentes: Typhlopidae)

Natural history data are important for a better understanding of distinct aspects of snake biology, and this information in scarce on Scolecophidia. Here we focus on sexual maturity and sexual dimorphism within a population of Amerotyphlops brongersmianus from the Restinga de Jurubatiba National Park, Rio de Janeiro state, Brazil. The smallest sexually active male and female showed snout-vent length of 117.5 and 158.4 mm, respectively. Females had statistically significant larger body and head length sizes, whereas males had longer tails. Juveniles showed no sexual dimorphism for any analyzed feature. Secondary vitellogenic follicles had a more opaque, yellowish/darker aspect, being larger than 3.5 mm. We reinforce that in addition to traditional features for determining sexual maturity, morphological and histological characteristics of kidneys should be evaluated in males, as well as the morphology of the infundibulum in females. Histological data show development of seminiferous tubules and presence of spermatozoa in males, and infundibulum receptacles and uterine glands in females as a sign of sexual maturity. This type of information is essential for a more accurate description of data on sexual maturity, allowing access to information on the development of reproductive structures that are not available macroscopically.

How Does Oocyte Uptake Occur? A Macroscopic Study of the Ovarian and Oviductal Modifications for Egg Capture in the Coral-Snake Micrurus corallinus.

The processes of follicular development, ovulation, egg capture, and egg transport vary among vertebrates. Therefore, analysis of the reproductive tract of several lineages is needed for understanding the evolutionary changes of the reproductive system. In turtles, the ovulated eggs are released into the coelomic cavity and taken up by one of the two oviducts, a phenomenon called extrauterine migration of eggs. However, the process of egg uptake in lizards is different. The egg is ovulated directly into the infundibulum, and oocyte uptake by the contralateral oviduct rarely occurs. The same pattern has been hypothesized to occur in snakes. To test this hypothesis, we analyzed the reproductive tract of female Micrurus corallinus preserved in zoological collections. We examined the anatomical characteristics of the infundibulum and ovary and compared the reproductive output between the ovaries to verify the mechanism of egg capture and the anatomical viability of extrauterine migration of eggs. The reproductive output of the right ovary was higher than that of the left ovary, and the higher number of eggs in the right oviduct is due exclusively to the production of the ipsilateral ovary. Several anatomical features prevent extrauterine migration of eggs, including the asymmetry of the reproductive system, the arrangement of the ovarian follicles in a single row, and the formation of a wrapping around the ovary and infundibulum by the visceral pleuroperitoneum membrane (preventing against ectopic eggs). Therefore, the hypothesis of egg capture by the contralateral oviduct is anatomically infeasible in M. corallinus and possibly in other snakes. Anat Rec, 301:1936-1943, 2018. © 2018 Wiley Periodicals, Inc.

Peering into the unknown world of amphisbaenians (Squamata, Amphisbaenia): A summary of the life history of Amphisbaena alba

Capturing data on the life of fossorial vertebrates is difficult since access to the subterranean environment is made unfeasible by its density and opacity. Collecting specimens is only possible through excavation work, causing damage or even death to the specimens. Due to the obstacles of in situ studies, the scarce information comes from reports obtained indirectly, mainly through specimens preserved in museums. Considering the adaptations to fossoriality, investments in studying these groups could be very enlightening since they would contribute enormously to the knowledge of the evolutionary strategies developed throughout the colonisation of the subterranean world. Amphisbaena alba is the species of Amphisbaenia with the broadest geographic distribution in the world. It occupies virtually all countries in South America except for Chile and southern Argentina. This study, carried out over the last 36 years, aims to provide data on the biology and behaviour of A. alba in captivity and in the field. Our main objective is to provide subsidies to expand the knowledge of the life history of this species and, by extension, of amphisbaenians in general.

Ultrasonographic and radiographic evaluation of gestationin golden lanceheads (Bothrops insularis) in ex situbreeding programs

The golden lancehead (Bothrops insularis) is a critically endangered snake endemic to Queimada Grande Island, southeastern Brazil. Captive breeding programs are vital for the conservation of this species. This study evaluates the gestation of two females golden lanceheads using radiography and ultrasonography. The first female was collected on the island while reproductively active (late vitellogenesis or early pregnancy) and kept in captivity. This female gave birth to five neonates after an 8-month gestation period, which is longer than estimates based on specimens preserved in museums. The second female copulated in captivity in July and probably ovulated in October. In this case, no embryonic development was detected, and the female deposited 14 undeveloped eggs approximately 6 months after ovulation.

Reproductive cycles of neotropical boid snakes evaluated by ultrasound

Snakes have increasingly been bred as pets around the world. Few studies have addressed the reproduction of boid snakes, and no study has addressed their reproductive cycles in captivity. Thus, this paper describes the reproductive aspects of Brazilian boids in captivity. We used ultrasonography to characterize the reproductive cycle of four boid species in captivity in the Southern Hemisphere: the anaconda (Eunectes murinus), the red-tailed boa (Boa constrictor constrictor), the Amazon tree boa (Corallus hortulanus), and the rainbow boa (Epicrates cenchria). Nonvitellogenic follicles occurred from January to December in anaconda and red-tailed boa and for a shorter period from September to February in Amazon tree boa and from January to May in rainbow boa. Vitellogenesis occurred from late June to late March in E. murinus in year-round (12 months), from March to March in Amazon tree boa, from late September to late March in red-tailed boa, and from late March to late September in rainbow boa. Mating occurred from late March to late September in red-tailed boa and rainbow boa and from late September to late March in Amazon tree boa. No mating was observed in anacondas, but a female probably underwent parthenogenesis. Births occurred in July in anaconda and in March to July in Amazon tree boa and from December to March in red-tailed boa and rainbow boa. In males, increases in testicular size were associated with the mating season. Ultrasonography proved to be a safe and noninvasive technique to study the reproductive cycle of giant snakes in captivity.